Redefining the role of the endogenous XAP2 and C-terminal hsp70-interacting protein on the endogenous Ah receptors expressed in mouse and rat cell lines.

Studies using transient expression systems have implicated the XAP2 protein in the control of aryl hydrocarbon receptor (AHR) stability and subcellular location. Thus, studies were performed in cell lines that expressed endogenous rat or mouse Ah(b-1) (C57BL/6) or Ah(b-2) (C3H) AHRs with similar levels of endogenous XAP2. Unliganded rat and mouse Ah(b-2) receptor complexes associated with reduced levels of XAP2 and exhibited dynamic nucleocytoplasmic shuttling in comparison with Ah(b-1) receptors. Rat and mouse Ah(b-2) receptors also exhibited a greater magnitude of ligand-induced degradation than Ah(b-1) receptors. Small interfering RNA reduction of endogenous XAP2 by >80% had minimal impact on the level of Ah(b-2) receptors but resulted in a 25-30% reduction of Ah(b-1) receptors. XAP2 reduction resulted in increased susceptibility of the Ah(b-1) receptor to ligand-induced degradation yet produced higher levels of endogenous CYP1A1 induction. Stable expression of the Ah(b-2) receptor in the C57BL/6 background resulted in a protein with reduced association with XAP2, dynamic nucleocytoplasmic shuttling, and increased levels of ligand-induced degradation. Small interfering RNA reduction of endogenous XAP2 in a C-terminal hsp70-interacting protein knockout mouse cell line, exhibited a 25-30% reduction in the level of endogenous Ah(b-1) AHR and showed high levels of ligand-induced degradation. Thus, endogenous XAP2 exerts a negative function on a small fraction of the endogenous Ah(b-1) receptor complex but appears to have a minimal impact on endogenous rat or Ah(b-2) receptors. This implies that the analysis of the AHR-mediated signaling via rat and mouse Ah(b-2) receptors may better represent the physiology of this signal transduction pathway.

The hepatitis B virus X-associated protein, XAP2 (also termed ARA9 and AIP) was identified in 1996 by its interaction and inhibition of the hepatitis B virus X protein (1). Structurally, XAP2 shares sequence identity with the FKBP52 class of immunophilins that contain a tetratricopeptide repeat motif typical of proteins that interact with hsp90 (reviewed in Ref. 2). The function of the immunophilins is still an evolving process, but FKBP52 has been shown to participate in the subcellular trafficking of steroid hormone receptors and also influence their stability and transactivation potential (reviewed in Ref. 3). However, XAP2 is not a true immunophilin, since it does not bind to compounds such as FK506 and does not share full identity with other immunophilins in the consensus tetratricopeptide repeat motif domain residues (2,4). Inter-est in XAP2 in the context of Ah receptor (AHR) 2 signal transduction began when yeast two-hybrid screens and direct immunoprecipitation identified XAP2 as a component of the mouse AHR⅐hsp90 complex (5)(6)(7). Since this discovery, numerous studies have been performed to assess the function of XAP2 in AHR-mediated signaling. With few exceptions, the analysis of AHR and XAP2 interactions has been carried out in vitro or in transient expression systems utilizing the Ah b-1 receptor that is expressed in the C57BL/6 strain of mouse (8). Some consistent conclusions from many of the studies are that overexpression of XAP2 leads to an increase in the level of cellular AHR protein and increases the level of AHR-mediated gene induction (5, 6, 9 -12). The increased levels of cellular AHR protein have been proposed to be the result of reduced ubiquitylation of the AHR through competition with the C-terminal hsp70-interacting protein (CHIP) (13,14). However, these conclusions are based on the analysis of a constitutively nuclear form of the AHR that exhibits a high level of ligand-independent degradation (15).
Another common finding among the various studies is that XAP2 expression can impact the subcellular localization of the AHR and appears to prevent dynamic nucleocytoplasmic shuttling (13, 16 -18). XAP2 has been hypothesized to mediate the nucleocytoplasmic shuttling of the AHR⅐hsp90 complex by altering its binding to importin ␤ (19). However, this finding may not be universal, since recent studies suggest that the expression of XAP2 has little impact on the subcellular localization or expression level of the human AHR in transient expression assays (20). In addition, an Ah b-1 receptor carrying an alanine mutation at tyrosine 408 does not associate with XAP2 and shows increased levels of transactivation of reporter genes (21). Thus, the overexpression of XAP2 has been shown to both increase and decrease the ligand-mediated activity of various reporter genes and impact the stability and localization of exogenously expressed AHRs from different species in distinct ways. These contradictory results highlight the need to assess the function of endogenous XAP2 on the stability and localization of the endogenous AHRs from different species. are identical to those described previously (22,23). Antibodies to XAP2 (Novus, Littleton, CO), hsp90 and p23 (Affinity BioReagents, Golden, CO), actin (Sigma), CHIP (Calbiochem), and rat P4501A1 (Chemicon International, Temecula, CA) were purchased from commercial sources. For Western blot analysis, goat anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase (GAR-HRP and GAM-HRP) were utilized. For immunohistochemical studies, goat anti-rabbit IgG conjugated to rhodamine (GAR-rhodamine) was used. Both of these reagents were purchased from Jackson Immunoresearch (West Grove, PA).
Cells and Growth Conditions-Wild type Hepa-1c1c7 (Hepa-1), and type I (LA-I) Hepa-1 variants were a generous gift from Dr. James Whitlock, Jr. (Department of Pharmacology, Stanford University). Mouse C2C12 myoblasts, mouse 10T1/2 embryonic fibroblasts, and rat A7H smooth muscle cells were obtained from ATCC (Manassas, VA). Transformed lung fibroblasts derived from CHIP Ϫ/Ϫ knockout and ϩ/ϩ mice were a generous gift from Cam Patterson (University of North Carolina, Chapel Hill, NC). Hepa-1, A7, and CHIP Ϫ/Ϫ cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. C2C12 cells were propagated in minimal essential medium supplemented with 10% fetal bovine serum. 10T1/2 cells were propagated in basal medium Eagle supplemented with 10% heat-inactivated fetal bovine serum. All cells were passaged at 1-week intervals and used in experiments during a 3-month period at ϳ70 -90% confluence. For treatment regimens, stated concentrations of TCDD, GA, or LMB were administered directly into growth media for the indicated incubation times.
Preparation of Total Cell Lysates-Following treatment, cell monolayers were washed twice with phosphate-buffered saline and detached from plates by trypsinization (0.05% trypsin, 0.5 mM EDTA). Cell pellets were washed with phosphate-buffered saline and sonicated directly in 75-150 l of ice-cold lysis buffer for 12 s. Lysates were immediately heated for 3 min at 100°C and then sonicated an additional 5 s. Samples were stored at Ϫ20°C until analysis.
Preparation of Cytosol and Immunoprecipitation-Cells were harvested by trypsinization as described above and disrupted by vortexing in radioimmune precipitation buffer supplemented with phenylmethylsulfonyl fluoride (10 mM), and aprotinin (10 M). Cytosol was generated by centrifugation at 30,000 ϫ g and stored at Ϫ80°C. Protein concentrations were determined using the Commassie Plus assay (Pierce) with bovine serum albumin as the standard. For immunoprecipitation, 600 -800 g of cytosol was precipitated in radioimmune precipitation buffer supplemented with bovine serum albumin (20 g/ml), DL-histidine (20 mM), 5 g of specific or preimmune IgG and 25 l of Protein A/G-agarose (Pierce) for 2.5 h at 4°C. Pellets were washed with TTBS containing sodium molybdate (20 mM) three times for 5 min at 4°C and protein and eluted by boiling in 30 l of SDS sample buffer. Samples were centrifuged at 30,000 ϫ g, and 15 l of the supernatant was resolved by discontinuous polyacrylamide slab gels (SDS-PAGE) and blotted to nitrocellulose.
Western Blot Analysis and Quantification of Protein-Equal amounts of protein were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose. Immunochemical staining was carried out with varying concentrations of primary antibody (see figure legends) in BLOTTO buffer supplemented with DL-histidine (20 mM) for 1-2 h at 22°C. Blots were washed with three changes of TTBSϩ for a total of 45 min. The blot was incubated in BLOTTO buffer containing a 1:10,000 dilution of appropriate peroxidase-conjugated secondary antibody for 1 h at 22°C and washed in three changes of TTBSϩ as above. Prior to detection, the blots were rinsed in phosphate-buffered saline. Bands were visualized with the enhanced chemiluminescence (ECL) kit as specified by the manufacturer (GE/Healthcare, Piscataway, NJ). Multiple exposures of each set of samples were produced. The relative concentration of target protein was determined by computer analysis of the autoradiographs as detailed previously (23)(24)(25).
Immunofluorescence Staining and Microscopy-All immunocytochemical procedures (cell plating, fixation, and staining) were carried out as previously described (22)(23)(24). Cells were observed on an Olympus IX70 microscope. On average, 15-20 fields (5-20 cells each) were evaluated on each coverslip, and 3-4 fields were photographed with a digital camera at the same exposure time to generate the raw data. Nuclear fluorescence intensities of 25-50 cells in three distinct fields of view, were obtained using MicroSuite image analysis software (Olympus America Inc.).
Generation of Stable Cell Lines-cDNA to the Ah b-2 receptor was a generous gift from Alan Poland (NIOSH). The Ah b-2 cDNA was ligated into the pLNCX2 retroviral expression vector (Clontech, Mountain View, CA), and virus particles used to infect the LA-I Hepa-1 variant cell line as described previously (26). For all experiments described in this report, three independent stable cell lines were evaluated. Parental LA-I cells and stable cell lines expressing murine C57Bl/6 AHR (AH WT ) have been described previously (26). All cells were maintained in selective medium during propagation, but G418 was not present in the medium when cells were plated for experiments.
RNA Interference-Annealed small interfering RNA (siRNA) complexes containing 21-bp regions of identity to regions of the murine XAP2 or an annealed 21-bp RNA to sequence not present in the mouse genome (siRNA control) were purchased from Ambion (Austin, TX). siRNA (final concentration of 50 -100 nM) was transfected into 35-mm dishes containing 1-2 ϫ 10 5 /cells using Lipofectamine TM reagent (Invitrogen). 36 -48 h after transfection, cells were treated as detailed in the figure legends, and total cell lysates were harvested for Western blotting. The efficiency of siRNA gene knockdown of XAP2 was determined by Western blotting. Transfection efficiency was monitored by microscopy using fluorescein isothiocyanate-labeled RNA (Clontech). In general, transfection efficiency in all experiments was Ͼ85%.
Statistical Analysis-Target protein bands were normalized to internal standards (actin), and the normalized densitometry units were compared by analysis of variance and Tukey-Kramer multiple comparison tests using InStat software (GraphPad Software Inc., San Diego, CA). Results are presented as mean Ϯ S.E. A probability value of Ͻ0.05 was considered significant.

RESULTS
The Endogenous Rat and Mouse Ah b-2 Receptors Are Not Associated with High Levels of Endogenous XAP2-It has been proposed that XAP2 functions in AHR-mediated signal transduction by influencing the stability and shuttling behavior of the AHR (5-7, 9 -19). However, these conclusions have been based primarily on the analysis of AHR and XAP2 in transient transfection systems. There is minimal information on the function of endogenous XAP2 especially as it relates to interac-tions with AHRs from species other than C57BL/6. To begin to address XAP2 function across species lines, studies were carried out on mouse Hepa-1c1c7 cells (expressing the C57BL/6 Ah b-1 receptor), mouse C2C12 myoblasts (expressing the Ah b-2 receptor), and rat smooth muscle cells (A7). The choice of these cells was based on our ability to detect endogenous AHR in fixed cells and by Western analysis (23,24,27). Fig. 1A shows a Western blot of total cell lysates stained for AHR, hsp90, XAP2, p23, and actin. As expected, the rat and mouse Ah b-2 receptors migrated at ϳ105 kDa and showed variation in the level of expression when compared with the Ah b-1 receptor (23). Importantly, the level of endogenous hsp90, p23, and XAP protein was equal between all the cell lines (Fig. 1A). Thus, due to the reduced level of endogenous AHR in the C2C12 and A7 cells (as compared with Hepa-1), the ratio of AHR/XAP2 in these cells would be expected to be smaller than that in Hepa-1 cells.
Since the association of XAP2 with the AHR has been predominantly carried out using in vitro expression systems and transient transfection assays, it was important to determine whether there were differences in the actual association of the endogenous AHRs with XAP2. Cytosol was produced from Hepa-1, A7, and C2C12 cells, and AHR was immunoprecipitated as detailed under "Experimental Procedures." The precipitated proteins were then analyzed by Western blotting, and a representative experiment is presented in Fig. 1B. The XAP2 protein was effectively co-precipitated by a specific AHR IgG from Hepa-1 cells. In contrast, the level of XAP2 co-precipitated from the C2C12 and A7 cells was greatly reduced, although the overall level of AHR precipitated from the cells were similar to that from the Hepa-1. Indeed over three experiments, the amount of XAP2 co-precipitated from A7 cells averaged 15 Ϯ 7% of the level co-precipitated from Hepa-1, whereas the level co-precipitated from C2C12 averaged 19 Ϯ 6%. Since previous studies suggest that the Ah b-2 receptor complex is less stable than the Ah b-1 receptor complex (22, 28 -30), sodium molybdate was used in all experiments to stabilize the AHR⅐hsp90 interactions (31,32). In addition, to control for the possibility that salt or nonionic detergent could affect the XAP2 association, additional precipitations were carried out with reduced amounts of NaCl (50 mM) and Nonidet P-40 (0.05%). The results were unchanged from those presented in Fig. 1B. These results represent the first studies to evaluate the association of endogenous AHRs from different species with endogenous XAP2. The key finding is that the unliganded AHR complex in the A7 and C2C12 is associated with 80 -90% less XAP2 than the Ah b-1 receptor in Hepa-1 cells. Interestingly, recent studies suggest that the human AHR is associated with reduced levels of XAP2 when transiently expressed in COS cells (20).
The Endogenous Rat and Mouse Ah b-2 but Not the Ah b-1 Receptor Shows Dynamic Nucleocytoplasmic Shuttling-The endogenous Ah b-1 receptor is predominantly cytoplasmic in Hepa-1 cells when evaluated by epifluorescence or confocal microscopy (22,24). 2 However, exogenous expression of the Ah b-1 receptor in HeLa or COS cells is nuclear unless XAP2 is also expressed in the cells (13, 16 -19). Thus, it has been hypothesized that XAP2 inhibits nucleocytoplasmic shuttling of the AHR complex. Since the endogenous AHRs in Hepa-1, A7, and C2C12 cells were associated with different levels of XAP2, it was pertinent to assess the dynamics of these AHRs in living cells. Consistent with previous studies, the endogenous Ah b-1 receptor in untreated Hepa-1 exhibits a predominantly cytoplasmic location that becomes strongly nuclear following exposure to ligand (Fig. 2). However, when nuclear export is inhibited by LMB, there is no observable change in the subcellular location of the endogenous Ah b-1 receptor. This finding was reproducible even after exposure of cells to 50 nM LMB for 9 h, and it is consistent with previous reports in Hepa-1 cells (27,33). In contrast, the endogenous AHRs expressed in C2C12, A7, and 10T1/2 embryonic fibroblasts (expressing the Ah b-2 receptor), exhibit a nuclear and cytoplasmic localization in untreated cells that becomes predominantly nuclear after a 60-min exposure to TCDD (Fig. 2). Importantly, exposure of each of these cells to LMB for 2 or 4 h results in a dramatic accumulation of the AHR in the nuclear compartment. In A7 cells, the nuclear shift is complete within 90 min of LMB exposure, whereas in the C2C12 and 10T cells, maximal nuclear location occurs between 2 and 3 h after LMB exposure. The LMB-induced accumulation of nuclear AHR suggests that the endogenous rat and mouse Ah b-2 receptors are actively shuttling between the cytoplasm and nucleus with a periodicity of 90 -180 min. LMB-induced nuclear localization of the endogenous AHR was also observed in the MCF7 human breast cancer line and has been shown for the endogenous zebrafish AHR2 in the ZFL cell line (34). 3 These results indicate that there exist strain and species differences in the nucleocytoplasmic shuttling of the endogenous AHR and that increased shuttling correlates to a reduced level of XAP2 associated with the AHR complex.  Reactivity was visualized by ECL with GAR-HRP or GAM-HRP IgG (1:10,000). B, cytosol was prepared from Hepa-1, A7, or C2C12 cells as detailed under "Experimental Procedures." 800 g of cytosol was precipitated with either affinity-pure A1-A IgG (5 g) or affinity-pure preimmune rabbit IgG (5 g) along with Protein A/G-agarose (25 l) for 2.5 h at 4°C with rocking. Pellets were washed three times for 5 min each with TTBS supplemented with sodium molybdate (20 mM) and then boiled in 30 l of SDS sample buffer. 15 g of cytosol (input) or 15 l of the eluted protein were resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml) or XAP2 mouse IgG 1 (1:750). Reactivity was visualized by ECL with GAR-HRP or GAM-HRP IgG (1:10,000). He, Hepa-1; C2, C2C12; Ah, precipitated with A1-A IgG; Pi, precipitated with preimmune IgG. The numbers under the XAP2 blot represent the percentage of XAP in relation to the level in Hepa-1 (100%).

The Endogenous Rat and Mouse Ah b-2 Receptors Show Distinct Profiles of Ligand-induced Degradation in Comparison with the Ah b-1
Receptors-The next series of studies were designed to evaluate the stability of the endogenous AHR following ligand exposure. Hepa-1, C2C12, and A7 cells were treated with TCDD for 0 -120 min, and the level of AHR was evaluated in total cell lysates by quantitative Western blotting. A representative experiment is shown in Fig. 3. Treatment of C2C12 or A7 cells with TCDD results in a dramatic reduction of cellular AHR protein by the 120-min time point. In C2C12 cells, AHR protein was not detectable at the 90-min time point, whereas the level of AHR in A7 cells decreased by ϳ88% of control levels by the 120-min time point. Consistent with previous reports, the AHR was also reduced by greater than 90% following 120 min of TCDD exposure in the 10T cell line (22,30). 3 In contrast, TCDD-induced degradation of the Ah b-1 receptor exhibited a shallow degradation profile and was only reduced by 30 -40% after 120 min. These results indicate that the endogenous Ah b-2 and rat receptors are degraded to near completion within 2 h of ligand exposure and highlight another striking difference in the response of these AHRs to the Ah b-1 receptor. Since previous studies suggest that 15-25% of the endogenous Ah b-1 receptors in Hepa-1 cells are not degraded even after 4 -16 h of TCDD exposure (24,26,27,(35)(36)(37), it was of interest to determine whether this population was still associated with XAP2. Cytosols prepared from Hepa-1 cells were treated with TCDD or vehicle for 4 h, and the AHR was immunoprecipitated as described under "Experimental Procedures." The results are shown in Fig. 3C. Although the endogenous Ah b-1 receptors were reduced by Ͼ80% following TCDD exposure, there were no changes in the level of endogenous XAP2, and the precipitated receptors were clearly associated with XAP2. It is noteworthy that the Ah b-1 receptors that are not degraded are still associated with XAP2.
Reduction of Endogenous XAP2 with siRNA Results in Reduced Levels of Endogenous Ah b-1 but Not Ah b-2 Receptors-To directly investigate the impact of XAP2 on the stability of the endogenous AHR, siRNAs were used to reduce the endogenous level of XAP2 in 10T, C2C12, and Hepa-1 cells. For these studies, siRNA specific to mouse XAP2 was transfected into cells, and the level of endogenous AHR and XAP2 was evaluated after 36 -48 h. To control for the cellular effects of siRNA transfection, siRNAs with no sequence identity to the mouse genome were included in all studies. A representative set of experiments is shown in Fig. 4. In all studies, the level of endogenous XAP2 expression FIGURE 2. Subcellular localization of AHR in Hepa-1, A7, C2C12, and 10T1/2 cells exposed to TCDD or LMB. Cells were grown on glass coverslips exposed to the compounds detailed below and then fixed as detailed previously (22)(23)(24). Coverslips were incubated with A-1 IgG (1.0 g/ml) and visualized with GAR-rhodamine IgG (1:400). A-D, Hepa-1 cells exposed to methanol (0.5%) for 4 h (A), TCDD (2 nM) for 1 h (B), LMB (20 nM) for 2 h (C), or LMB for 4 h (D). E-H, C2C12 cells exposed to methanol (0.5%) for 4 h (E), TCDD (2 nM) for 1 h (F), LMB (20 nM) for 2 h (G), or LMB for 4 h (H). Panels I-L, A7 cells exposed to methanol (0.5%) for 4 h (I), TCDD (2 nM) for 1 h (J), LMB (20 nM) for 2 h (K), LMB for 4 h (L). M-P, 10T1/2 cells exposed to methanol (0.5%) for 4 h (M), TCDD (2 nM) for 1 h (N), LMB (20 nM) for 2 h (O), or LMB for 4 h (P). Each set of four panels was exposed for identical times. Bar (A), 10 m.
was reduced by 80 -90% as determined by quantitative Western blotting. Importantly, the level of endogenous Ah b-1 receptor was decreased by 26 Ϯ 5% in Hepa-1 cells with reduced XAP2 (Fig. 4D). These findings were consistent and reproducible over four experiments. In contrast, the reduction of XAP2 in 10T or C2C12 cells had no measurable impact on the endogenous concentration of the Ah b-2 receptor (Fig. 4). The impact of XAP2 reduction in A7 cells could not be evaluated as transfection efficiency was not sufficient to reduce XAP2 levels below 50%. Collectively, these results support a role of endogenous XAP2 in the stability of a portion of the unliganded Ah b-1 receptor and illustrate another distinction between the Ah b-1 and Ah b-2 receptors.
Reduction of Endogenous XAP2 with siRNA Results in Increased Levels of Ligand-induced Degradation of the AHR and Enhanced Levels of CYP1A1 Induction in Hepa-1 Cells-The reduction of endogenous Ah b-1 receptor by 25-30% is intriguing, since it has been hypothesized that there are distinct pools of AHR⅐hsp90 complexes in Hepa-1 cells (12), and previous studies have consistently shown that ϳ20 -25% of the endogenous AHR pool remains in Hepa-1 cells after ligand exposure (Fig. 3) (24, 26, 27). Therefore, it was of interest to evaluate the ligandinduced degradation of the AHR in Hepa-1 cells with reduced levels of XAP2. Hepa-1 cells were transfected with control or XAP2 siRNA as described and allowed to recover for 48 h. Cells were then treated with TCDD for 0 -6 h, total cell lysates were produced, and the level of XAP2, AHR, actin, and CYP1A1 was evaluated by quantitative Western blotting. A representative study is shown in Figs. 5 and 6. As observed in Fig. 4, XAP2 was significantly reduced (85%), and there was a corresponding reduction in the level of endogenous AHR (Fig. 5). Importantly, in cells with reduced levels of XAP2, the endogenous Ah b-1 receptor was more susceptible to TCDD-induced degradation (Fig. 5). Indeed, the Ah b-1 AHR was reduced by 52% at the 2-h time point compared with 30% in cells transfected with control siRNA. The overall level of AHR degradation at the 6-h time point was also higher in cells with reduced XAP2. Thus, these results suggest that the association of XAP2 with the Ah b-1 receptor complex impacts the time course and magnitude of ligand-induced degradation To assess the impact of reduced XAP2 on induction of the endogenous CYP1A1 gene, the identical samples shown in Fig. 5, were evaluated for the expression of CYP1A1 protein (Fig. 6). Importantly, reduction in XAP2 resulted in an increase in the endogenous CYP1A1 protein detected after 2, 4, and 6 h of TCDD exposure. At each time point, the level of CYP1A1 protein was increased by ϳ26% compared with the levels in cells that did not have reduced XAP2. These findings support the hypothesis that endogenous XAP2 acts to repress a population of AHRs from the induction of the endogenous CYP1A1 gene. This observation would be expected if the same populations of AHR that are resistant to degradation were also incapable of mediating gene regulation.
Ah b-2 Receptors Expressed in the Hepa-1 Background Are Associated with Reduced Levels of XAP2 and Exhibit Higher Levels of Ligand-in- FIGURE 3. Western blot analysis of AHR in Hepa-1, A7, and C2C12 cells exposed to TCDD. A, Hepa-1, A7, and C2C12 cells were exposed to Me 2 SO (0.1%) for 2 h or TCDD (2 nM) for the indicated times. Equal amounts of total cell lysates were then resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml) and ␤-actin IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP IgG (1:10,000), and bands were quantified and normalized as detailed (23)(24)(25). duced Degradation-To further assess the differences in stability and shuttling observed between the Ah b-1 and Ah b-2 receptors, retroviral infection was used to generate stable cell lines expressing each AHR in the same genetic background. The Hepa-1 LA-I variant line was used for these studies, since the cells express low levels of endogenous AHR (38). This cell line has also been used to generate stable cell lines expressing physiological levels of mouse AHR (26). Fig. 7A shows the expression of AHR, hsp90, XAP2, and actin in the parental LA-I variant, wild type Hepa-1 (He), and the stable cells lines designated AH WT (expressing the Ah b-1 receptor) (see also Ref. 26) and AH b2 (expressing the Ah b-2 receptor). Importantly, the level of AHR expressed in each of the stable cell lines approximates that of the endogenous AHR in wild type Hepa-1 cells, and the levels of hsp90 and XAP2 are also similar. To assess the composition of the unliganded AHR complex, cytosol was generated from two independent AH b2 cell lines as well as the AH WT line and immunoprecipitated with antibodies specific to the AHR. As shown for the endogenous Ah b-1 receptor in Hepa-1 cells, immunoprecipitation of the AHR from the AH WT cells co-precipitated a high level of XAP2 (Fig.  7B). In contrast, as shown for the endogenous rat and Ah b-2 receptors, immunoprecipitation of the AHR from both of the AH b2 cell lines pulled down significantly less amounts of endogenous XAP2 (Fig. 7B). Over several experiments, the average amount of XAP2 co-precipitated from the AH b2 cells was ϳ21 Ϯ 8% of the level co-precipitated from AH WT . Thus, the species of receptor and not the cellular context of its expression determine the association with XAP2.
Because the AHRs expressed in the A7 and C2C12 cells exhibited increased susceptibility to TCDD-induced degradation, it was pertinent to assess the degradation of the AHR in the AH b2 cell line. A representative Western blot experiment is shown in Fig. 7C, and the quantified results are presented in Fig. 7D. TCDD-induced degradation in the AH b2 line is more rapid and proceeds to a greater magnitude than that in the AH WT . Indeed, the Ah b-2 receptor is reduced by 52% after 2 h and Ͼ85% after 6 h of TCDD exposure compared with 28 and 74% for the Ah b-1 receptor. Overall, the profile of AHR degradation in the AH b2 line is more representative of that observed in the A7, C2C12, and Hepa-1 line with reduced XAP2 than the WT Hepa-1 line (compare Figs. 3B, 5C, and 7D).
To assess any differences in the ability of the Ah b-1 or Ah b-2 receptors to induce CYP1A1, AH b2 and AH WT cells were treated with TCDD for 0 -6 h, and the level of CYP1A1 protein was evaluated by Western blotting (Fig. 8). Fig. 8A shows that CYP1A1 was detectable within 2 h of TCDD exposure in both cells lines and increased proportionally over time. To determine whether there were any quantitative differences in the induction of CYP1A1 protein in the two cell lines, cells were treated with TCDD for 16 h, and identical levels of total cell lysate were evaluated by quantitative Western blotting. To show that the induction of CYP1A1 protein was due to the expression of the integrated AHRs, the parental LA-I cell line was also evaluated over the same time frame. Surprisingly, the level of CYP1A1 protein induced after 16 h was not statistically different between the AH WT and AH b2 cell lines although the Ah b-2 receptor was associated with less XAP2 and degraded with a more rapid profile (Fig. 8, B and C). Thus, in these model systems, a reduction in the level of XAP2 in the core AHR complex did not appear to impact induction of the endogenous CYP1A1 gene.
Ah b-2 AHR Expressed in the Hepa-1 Background Exhibits Nucleocytoplasmic Shuttling-The endogenous Ah b-2 receptor expressed in C2C12 and 10T cells shows clear nucleocytoplasmic shuttling in comparison with the Ah b-1 receptor expressed in the Hepa-1 cell line (Fig. 2). Since each of these cells represents a different genetic background, the lack of shuttling in the Hepa-1 could be related to this background or be specific to the AHR expressed in the cell. To evaluate this question, AH WT and AH b2 cells were propagated on glass coverslips, exposed to TCDD or LMB, and then fixed and stained for AHR protein. As expected, the location of the Ah b-1 receptor in the AH WT cells was predominantly cytoplasmic and became nuclear following 1 h of TCDD treatment (Fig. 9). In addition, as observed in the wild type Hepa-1 lines, 4 h of LMB exposure did not influence the location of the Ah b-1 receptor. In contrast, the unliganded Ah b-2 receptor exhibited a nuclear and cytoplasmic location that became strongly nuclear in the presence of TCDD. Importantly, exposure of the AH b2 cells to LMB resulted in an

. Western blot analysis of AHR in
Hepa-1, C2C12, and 10T1/2 cells following siRNA knockdown of XAP2. Hepa-1, C2C12, and 10T1/2 cells were transfected with siRNA specific to XAP2 or control siRNA as detailed under "Experimental Procedures." 48 h later, total cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml) or XAP2 mouse IgG 1 (1:750) and actin IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP or GAM-HRP IgG (1:10,000), and protein bands were quantified and normalized as detailed (23)(24)(25). A, Hepa-1 and C2C12 cells. B, 10T1/2 cells. C, the levels of AHR protein in the control siRNA (siCon) and XAP2-specific siRNA (siXAP2) samples were divided by the corresponding level of actin, and the average Ϯ S.E. of the 6 -7 independent samples was plotted as a function of the control siRNA. *, statistically different from the control siRNA; p Ͻ 0.001. Reduction of endogenous XAP2 was Ͼ85% in all cell lines.
increased level of nuclear AHR in comparison with control-treated cells (Fig. 9, lower panels). Since the Ah b-2 receptor showed a nuclear location prior to LMB treatment, the change in nuclear fluorescence intensity was measured as detailed under "Experimental Procedures." The average level of fluorescence in control nuclei was 106 Ϯ 10 (n ϭ 100), whereas the LMB-treated nuclei averaged 165 Ϯ 12 (n ϭ 100). Thus, these results show that nucleocytoplasmic shuttling does occur in the Hepa-1 cell line but is dependent on the species of AHR that is expressed. When considered in the context of the results presented in Figs. 7 and 8, these findings show that the Ah b-2 receptor expressed in the C57BL/6 background exhibits distinct susceptibility to ligand exposure and nucleocytoplasmic shuttling when compared with the Ah b-1 receptor. If the association of the AHR with XAP2 in indeed involved in inhibiting nucleocytoplasmic shuttling (13, 16 -18), these findings further support the hypothesis that endogenous levels of XAP2 have minimal impact on Ah b-2 receptors.
Ligand-dependent and -independent Degradation of the Endogenous Ah b-1 Receptor Is Not Mediated by CHIP-It was next of interest to investigate the mechanism responsible for the reduction in the endogenous Ah b-1 receptor when XAP2 was reduced by siRNA. CHIP is an E3 ubiquitin ligase that has been implicated in the degradation of several target proteins that are associated with hsp90 (reviewed in Ref. 39). Recently, CHIP was shown to degrade a constitutively nuclear form of the Ah b-1 receptor (14). In addition, these same studies showed that the CHIP mediated degradation of the AHR was inhibited by the overexpression of XAP2. However, these studies were performed on a constitutively nuclear AHR that does not exhibit ligand-induced degradation and has a 2-h half-life (15). Therefore, it was pertinent to investigate whether there was a connection between the reduction in XAP2, CHIP, and the degradation of the endogenous Ah b-1 receptor.
Initial studies were performed on Hepa-1 and C2C12 cell lines using siRNA to CHIP. Although the endogenous CHIP protein could be reduced by 70% in each of the cell lines, there was no significant difference in the level of AHR expression or its degradation profile in the presence of TCDD or geldanamycin. 3 Therefore, there may have been sufficient CHIP enzyme remaining in the cells to mediate AHR degradation. In order to circumvent this problem, we utilized transformed FIGURE 5. Western blot analysis of AHR in Hepa-1 following siRNA knockdown of XAP2 and exposure to TCDD. Hepa-1 cells were transfected with siRNA specific to XAP2 (siXAP2) or control siRNA (siCON) as detailed under "Experimental Procedures." 48 h later, cells were treated with Me 2 SO (0.1%) for 6 h or TCDD (2 nM) for the indicated times. Total cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml), XAP2 mouse IgG 1 (1:750), and actin IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP or GAM-HRP IgG (1:10,000), and protein bands were quantified in identical exposures and normalized as detailed (23)(24)(25). A, samples from cells transfected with control siRNA or siRNA specific to XAP2. B, the time 0 controls from sample in A showing the level of XAP2 knockdown. C, the levels of AHR protein in the control siRNA and XAP2-specific siRNA samples were divided by the corresponding level of actin, and the average Ϯ S.E. of the three independent samples was plotted as normalized densitometry units. The error bars on some of the later time points are within the symbol used to show the data point. Note the 25% reduction in the level of AHR in the time 0 XAP2-specific siRNA samples compared with control siRNA. *, statistically different from the time-matched control siRNA; p Ͻ 0.001. Reduction of endogenous XAP2 was Ͼ85% in all cell lines. FIGURE 6. Western blot analysis of CYP1A1 protein in Hepa-1 following siRNA knockdown of XAP2 and exposure to TCDD. A, equal amounts of the identical samples described in Fig. 5 were resolved by SDS-PAGE, blotted, and stained with rat CYP1A1 IgG (1:10,000) and (1:750) actin IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000), and protein bands were quantified in identical exposures and normalized as detailed (23)(24)(25). Samples are shown from cells transfected with control siRNA (siCON) or siRNA specific to XAP2 (siXAP2). B, the levels of CYP1A1 protein in the control siRNA and XAP2-specific siRNA samples were divided by the corresponding level of actin, and the average Ϯ S.E. of the three independent samples was plotted as normalized densitometry units. *, statistically different from the control siRNA; p Ͻ 0.001. The level of XAP2 reduction in these sample is shown in Fig. 5, A and B. lung fibroblasts derived from CHIP knockout mice (40). The initial characterization of these cells is shown in Fig. 10. Importantly, both CHIP Ϫ/Ϫ and CHIP ϩ/ϩ cells express endogenous levels of AHR and exhibit no differences in the level of ARNT, XAP2, or hsp90. CHIP Ϫ/Ϫ cells appear homozygous for the Ah b-1 receptor, whereas CHIP ϩ/ϩ cells express the Ah b-1 and the Ah d receptor (from the DBA-derived embryonic stem cells). Quantification of the AHR expression in both cell lines showed that the total level of AHR protein was equal. To assess whether CHIP affected ligand-induced degradation, cells were treated with TCDD for 4 h, and the level of AHR in each line was evaluated by Western blotting (Fig. 10B). The level of ligand-induced degradation of the AHR was identical in both CHIP Ϫ/Ϫ and CHIP ϩ/ϩ cells. Thus, CHIP is not an enzyme used in the ligand-induced degradation of either the Ah b-1 or Ah d receptor.
The AHR is also degraded in a ligand-independent manner following exposure to geldanamycin (26, 35,41). GA binds to hsp90 and is thought to cause a conformational change in the AHR complex that predisposes it to degradation (41). GA exposure also results in displacement of XAP2 from AHR⅐hsp90 complex in vitro, although overexpression of XAP2 does not appear to protect the AHR from GA-induced degradation in transfected cells (13). To assess the role of CHIP in GA-induced degradation, cells were treated with GA for 2 h, and the level of AHR in each line was evaluated by Western blotting (Fig. 10C). The level of GA-induced degradation of the AHR was identical in both CHIP Ϫ/Ϫ and CHIP ϩ/ϩ cells. Thus, CHIP does not participate in the GA-induced degradation of either the Ah b-1 or Ah d receptor.
Finally, it was of interest to determine whether CHIP was involved in the loss of AHR that is observed following the reduction of XAP2 by siRNA (Figs. 4 and 5). For these studies, CHIP Ϫ/Ϫ cells were transfected with XAP2 siRNA and allowed to recover for 48 h. Cells were then exposed to TCDD for 4 h, and the levels of endogenous AHR, XAP2, and actin were evaluated by Western blotting (Fig. 11). Consistent with the results in the Hepa-1 cell line, the endogenous Ah b-1 receptor was decreased by 26% when XAP2 was reduced by siRNA. Collectively, these studies show that the CHIP E3 ubiquitin ligase is not involved in the degradation of the endogenous Ah b-1 receptor whether it is induced by TCDD, GA, or a reduced level of XAP2. In addition, the studies with the CHIP KO cells validate the CHIP siRNA work in the Hepa-1 and C2C12 cells and suggest that CHIP does not impact the level of the Ah b-2 receptor.

DISCUSSION
The role of XAP2 in the stability and nucleocytoplasmic shuttling behavior of the Ah b-1 receptor complex is based on overexpression studies that detected a higher level of cytoplasmic AHR protein and increased levels of AHR-mediated gene induction in the presence of exogenously expressed XAP2 in yeast or mammalian cells (5-7, 9 -11). A general assumption has been that the functions ascribed to XAP2 on  10,000). B, cytosol was prepared from AH WT (WT) or two independent AH b2 (b2) cell lines as detailed. 800 g of cytosol was precipitated with either affinity-pure A1-A IgG (5 g) or affinity-pure preimmune rabbit IgG (5 g) along with Protein A/G-agarose (25 l) for 2.5 h at 4°C with rocking. Pellets were washed three times for 5 min each with TTBS supplemented with sodium molybdate (20 mM) and then boiled in 30 l of SDS sample buffer. 15 g of cytosol (input) or 15 l of the eluted protein were resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml) or XAP2 mouse IgG 1 (1:750). Reactivity was visualized by ECL with GAR-HRP or GAM-HRP IgG (1:10,000). Ah, precipitated with A1-A IgG; Pi, precipitated with preimmune IgG. The numbers under the XAP2 blot represent the percentage of XAP in relation to the level in the AH WT cell line (100%). C, AH WT and AH b2 cells were treated with Me 2 SO (0.1%) for 6 h or TCDD (2 nM) for the indicated times. Total cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml) and actin IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000), and protein bands were quantified and normalized as detailed (23)(24)(25). D, the levels of AHR protein in the AH WT and AH b2 samples were divided by the corresponding level of actin, and the average Ϯ S.E. of the three independent samples was plotted as the percentage of time 0 control. *, statistically different from the control siRNA; p Ͻ 0.001.
the Ah b-1 receptor complex are universal for all other AHRs. The key findings of the current report challenge this view by showing that (i) very low levels of endogenous XAP2 associate with the endogenous rat and mouse Ah b-2 receptors, (ii) rat and mouse Ah b-2 receptors show dynamic nucleocytoplasmic shuttling in the presence of endogenous XAP2, (iii) endogenous XAP2 stabilizes only a small fraction of the Ah b-1 receptor pool, (iv) endogenous XAP2 has a negative impact on gene regulation mediated by the Ah b-1 receptor, and (v) endogenous XAP2 is not competing with the CHIP E3 ubiquitin ligase. Collectively, these results imply that XAP2 may only impact AHR-mediated signaling in the context of the Ah b-1 receptor and suggest that the analysis of the AHR-mediated signaling via rat and mouse Ah b-2 receptors may better represent the physiology of this signal transduction pathway.
So what is the role of XAP2 in AHR-mediated signaling? For the Ah b-1 receptor, the results support a role of XAP2 in the stabilization of a fraction of the Ah b-1 receptor pool, since there was only a modest reduction (25-30%) in endogenous AHR following XAP2 knockdown in Hepa-1 or lung fibroblast cells. This modest reduction of AHR is consistent with the finding that there may be multiple pools of endogenous AHR⅐hsp90 complexes in Hepa-1 cells and that less than 40% of these complexes are associated with XAP2 in vivo (12). Unlike the studies that utilized exogenous expression of AHR and XAP2 to assess the function of these proteins, the analysis of the endogenous proteins support a hypothesis that the populations of Ah b-1 receptor complexes that are associated with XAP2 are more resistant to ligand-induced degradation and to the formation of AHR⅐ARNT dimers capable of regulating genes. This hypothesis is supported by the finding that depletion of endogenous XAP2 by 85% results in increased susceptibility of the endogenous Ah b-1 receptor to ligand-induced degradation and a corresponding increase in the induction of CYP1A1 protein over control-treated cells. In addition, the 15-25% of Ah b-1 receptors that are resistant to ligand-induced degradation appear to be associated with XAP2. These results imply that the stabilization of the Ah b-1 receptor by XAP2 actually has a negative impact on AHRmediated signaling. Importantly, the ability of XAP2 to exert a negative function on gene regulation has recently been demonstrated in transient studies of the human AHR (20) and by analysis of a mutant Ah b-1 receptor that does not associate with XAP2 (21). In addition, it is important to note FIGURE 8. Western blot analysis of CYP1A1 protein in stable cell lines expressing Ah b-1 or Ah b-2 receptors following exposure to TCDD. A, AH WT and AH b2 cells were treated with Me 2 SO (0.1%) for 6 h or TCDD (2 nM) for the indicated times. Total cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE, blotted, and stained with rat CYP1A1 IgG (1.0 g/ml) and actin IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000). B, LA-I, AH WT , and AH b2 cells were treated with Me 2 SO (0.1%) or TCDD (2 nM) for 16 h. Total cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE, blotted, and stained with rat CYP1A1 IgG (1.0 g/ml) and actin IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000), and protein bands were quantified and normalized as detailed (23)(24)(25). C, the levels of CYP1A1 protein in the LA-I, AH WT , and AH b2 samples were divided by the corresponding level of actin, and the average Ϯ S.E. of the three independent samples was potted as normalized densitometry units. Con, average density of Me 2 SO-treated cells. FIGURE 9. Subcellular localization of AHR AH WT and AH b2 cells exposed to TCDD or LMB. Cells were grown on glass coverslips exposed to the compounds detailed below and then fixed as detailed previously (22)(23)(24). Coverslips were incubated with A-1 IgG (1.0 g/ml) and visualized with GAR-rhodamine IgG (1:400). A, B, D, and E, AH WT cells exposed to Me 2 SO (0.1%) (A) for 1 h, TCDD (2 nM) for 1 h (B), methanol (0.5%) for 4 h (D), or LMB (20 nM) for 4 h (E). F-I, AH b2 cells exposed to Me 2 SO (0.1%) for 1 h (F), TCDD (2 nM) for 1 h (G); methanol (0.5%) for 4 h (H), or LMB (20 nM) for 4 h (I). C, the parental LA-I cell line stained and photographed under the identical conditions to the AH WT and AH b2 cells. All panels were exposed for identical times. Bar (A), 10 m.
that the initial function of XAP2 was as a negative transcriptional regulator of the hepatitis B virus X protein (1).
The results presented in the current report also call into question the proposed mechanism whereby XAP2 stabilizes the endogenous Ah b-1 receptor complex through competition with the CHIP E3 ubiquitin ligase (14). Our results utilizing a cell line devoid of CHIP protein clearly establish that CHIP is not involved in the degradation of the endogenous Ah b-1 receptor whether it is induced by TCDD, GA, or a reduced level of XAP2. In addition, siRNA knockdown of CHIP in the C2C12 cell line did not impact the TCDD or GA induced degradation of the Ah b-2 receptor. 3 Therefore, these results indicate that the hypothesized competition between CHIP and XAP2 for binding to hsp90 is unlikely to be occurring in a physiological setting. The difference in results of this report from those of Lees et al. (14) are probably related to our analysis of an endogenous AHR that is functioning under physiological conditions, compared with the analysis of a tagged version of the AHR that is turning over rapidly. Since CHIP does not appear to impact AHR stability, the precise mechanism(s) that control ligand-induced or ligandindependent degradation is still not resolved. It is intriguing to consider that several studies have shown that the degradation of some nuclear receptors is correlated to transactivation, DNA binding, and the recruitment of co-activators to the promoter/enhancer region of responsive genes (42)(43)(44)(45)(46). The strong association of XAP2 with the Ah b-1 receptor complex (as compared with rat and Ah b-2 receptors), may slow down the transformation process and prevent a population of AHRs from interacting with ARNT, binding DNA, and being degraded. Interestingly, recent studies suggest that the Ah b-1 receptor remains associated at the CYP1A1 promoter during ligand stimulation, whereas the human AHR shows periods of association and dissociation at the CYP1A1 promoter and is suggestive of turnover (47,48). Future studies will be aimed at further analyzing gene regulation and degradation in the AH WT and AH b2 cell lines to see if there are differences that result from the reduced level of XAP2 association with Ah b-2 receptors.
Does endogenous XAP2 impact AHR-mediated signaling in species other than C57BL/6? In general, the results regarding XAP2 function on endogenous rat and Ah b-2 receptors are in strong contrast to those of the Ah b-1 receptor. When evaluated in the physiological stoichiometry of a cell, endogenous rat and mouse Ah b-2 receptors are associated with greatly reduced levels of endogenous XAP2. In addition, the cellular concentration of endogenous Ah b-2 receptors does not show a decrease when XAP2 is reduced by siRNA. Finally, endogenous rat and mouse Ah b-2 as well as stably expressed Ah b-2 receptor show dynamic nucleocytoplasmic shuttling in the presence of endogenous XAP2. The finding that the Ah b-2 and rat receptors are functioning in a distinct manner compared with the Ah b-1 receptor is not unprecedented. Indeed, when compared with human, rat and mouse AHRs from other strains, the Ah b-1 receptor expressed in the C57BL/6 mouse exhibits differences in biophysical stability (27,28,31), ligand-induced degradation (24,30), ligand binding affinity (32,49) and cycling at the CYP1A1 promoter (47,48). All of these characteristics are consistent with an AHR protein that possesses a level of stability not found in the receptors from other mouse strains, rats, and humans. The dramatic differences between the mouse Ah b-1 and Ah b-2 receptors are particularly striking, since the two receptors exhibit 99.1% amino acid sequence identity and differ only at amino acid 74 (Thr/Ser), 324 (Ile/Met), 348 (Leu/Phe), 471 (Leu/Pro) 533 (Ser/ Asn), and 758 (Ala/Thr). In addition, the Ah b-2 , rat, and human receptors all have a conserved tyrosine at amino acid 408 that has been implicated in the association of the Ah b-1 receptor with XAP2 (21). Thus, it is unlikely that the functional differences between the mouse Ah b-1 and Ah b-2 receptors are related to changes in the amino acid sequence over the first 805 amino acids. However, it is pertinent to note that the Ah b-1 receptor contains a point mutation that prematurely truncates the receptor at amino acid 805, whereas the mouse Ah b-2 , rat, and human AHRs all contain an additional 42-45 amino acids at their COOH terminus that have regions of 70% identity (8,29,50,51). This difference clearly distinguishes the Ah b-1 receptor from all other mammalian AHRs. Therefore, the analysis of AHR-mediated signaling via rat, human, and mouse Ah b-2 receptors may better represent the physiology of the AHR signal transduction pathway, since the mouse Ah b-1 receptor appears to be the outlier and is actually missing amino acid sequence that could be functionally significant. FIGURE 10. Western blot analysis of AHR in CHIP ؊/؊ or ؉/؉ cell lines following exposure to TCDD or GA. A, equal amounts of total cell lysates from the CHIP Ϫ/Ϫ or ϩ/ϩ cells were resolved by SDS-PAGE, blotted, and stained with A-1A rabbit IgG (1.0 g/ml), R-1 rabbit IgG (1 g/ml), ␤-actin rabbit IgG (1:1000), hsp90 rabbit IgG (1:500), XAP2 mouse IgG 1 (1:750), or CHIP rabbit IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP or GAM-HRP IgG (1:10,000). B, CHIP Ϫ/Ϫ or ϩ/ϩ cells were treated with TCDD (2 nM) for 4 h, and equal amounts of total lysates were resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml) and ␤-actin rabbit IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000). C, CHIP Ϫ/Ϫ or ϩ/ϩ cells were treated with GA (100 nM) for 2 h, and equal amounts of total lysates were resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml), CHIP IgG (1:1000), and ␤-actin rabbit IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000). Total cell lysates from Hepa-1 and C2C12 (C2) cells were included as molecular mass markers for the AHR. FIGURE 11. Western blot analysis of AHR in CHIP ؊/؊ cells following siRNA knockdown of XAP2. A, CHIP Ϫ/Ϫ cells were transfected with siRNA specific to XAP2 or control siRNA as detailed under "Experimental Procedures." 48 h later, total cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE, blotted, and stained with A-1A IgG (1.0 g/ml), XAP2 mouse IgG 1 (1:750), and actin IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000), and protein bands were quantified and normalized as detailed (23)(24)(25). B, the levels of AHR protein in the control siRNA and XAP2-specific siRNA samples were divided by the corresponding level of actin, and the average Ϯ S.E. of the four independent samples was plotted as a function of the control siRNA. The error bars on some of the later time points are within the symbol used to show the data point. *, statistically different from the time-matched control siRNA; p Ͻ 0.001. Reduction of endogenous XAP2 was Ͼ85%.