SIMPL Is a Tumor Necrosis Factor-specific Regulator of Nuclear Factor-κB Activity*

The IL-1 receptor-associated kinase (IRAK/mPLK) is linked to the regulation of nuclear factor-κB (NF-κB)-dependent gene expression. Here we describe a novel binding partner of IRAK/mPLK that we term SIMPL (signaling molecule that associates with the mouse pelle-like kinase). Overexpression of SIMPL leads to the activation of NF-κB-dependent promoters, and inactivation of SIMPL inhibits IRAK/mPLK as well as tumor necrosis factor receptor type I-induced NF-κB activity. Dominant inhibitory alleles of IκB kinase (IKKα or IKKβ) block the activation of NF-κB by IRAK/mPLK and SIMPL. Furthermore, SIMPL binds IRAK/mPLK and the IKKs in vitro and in vivo. In the presence of antisense mRNA to SIMPL, the physical association between IRAK/mPLK and IKKβ but not IRAK/mPLK and IKKα is greatly diminished. Moreover, dominant-negative SIMPL blocks IKKα- or IKKβ-induced NF-κB activity. These results lead us to propose a model in which SIMPL functions to regulate NF-κB activity by linking IRAK/mPLK to IKKβ/α-containing complexes.

The IL-1 receptor-associated kinase (IRAK/mPLK) is linked to the regulation of nuclear factor-B (NF-B)-dependent gene expression. Here we describe a novel binding partner of IRAK/mPLK that we term SIMPL (signaling molecule that associates with the mouse pelle-like kinase).

Overexpression of SIMPL leads to the activation of NF-B-dependent promoters, and inactivation of SIMPL inhibits IRAK/mPLK as well as tumor necrosis factor receptor type I-induced NF-B activity. Dominant inhibitory alleles of IB kinase (IKK␣ or IKK␤) block the activation of NF-B by IRAK/mPLK and SIMPL. Furthermore, SIMPL binds IRAK/mPLK and the IKKs in vitro and in vivo. In the presence of antisense mRNA to SIMPL, the physical association between IRAK/mPLK and IKK␤ but not IRAK/mPLK and IKK␣ is greatly diminished. Moreover, dominant-negative SIMPL blocks IKK␣-or IKK␤-induced NF-B activity. These results lead us to propose a model in which SIMPL functions to regulate NF-B activity by linking IRAK/mPLK to IKK␤/ ␣-containing complexes.
Tumor necrosis factor ␣ (TNF␣) 1 is a pro-inflammatory cytokine that functions as an activator of the innate immune response. Produced mainly by activated macrophages, TNF␣ can influence cell survival or cell death (for review see Ref. 1). TNF␣-mediated activation of nuclear factor-B (NF-B)dependent signaling is postulated to occur exclusively through the TNF receptor type I (TNF-RI). NF-B binds regulatory elements in the promoters of genes that encode mediators of the acute and chronic inflammatory response (for review see Refs. [2][3][4]. Prototypical NF-B is a heterodimer composed of a 50-kDa subunit (NF-B/p50) and a 65-kDa subunit (RelA/p65). NF-B activity is most likely regulated at two levels, cytoplasmic sequestration and direct removal from DNA. An additional protein family, inhibitors of B (IB), is involved in both regulatory processes (for review see Refs. 5 and 6).
The IL-1 receptor-associated kinase (IRAK) was identified as a protein associated with the type I IL-1 receptor (21), whereas the mouse homologue of IRAK, mouse pelle-like kinase (mPLK), was identified independently (22) in a cDNA library screened for kinases related to the Drosophila pelle kinase (23). We have demonstrated that IRAK/mPLK protein kinase activity is critical for TNF-RI signaling and placed mPLK/IRAK in a novel signal transduction pathway through which TNF-RI activates NF-B-dependent gene expression (24). Although the IRAK/mPLK protein may be required for IL-1-induced NF-B activity, IRAK/mPLK catalytic activity is not required for IL-1-dependent signaling (24 -26). In mPLK/IRAK null fibroblasts IL-1 as well as in TNF-induced NF-B, DNA binding activity is significantly attenuated (27). In this report we describe a novel signaling molecule that associates with the mouse pelle-like kinase (SIMPL) and demonstrate that SIMPL is required for TNF-RI-dependent activation of NF-B activity.
Northern Blot Analysis-Northern blots containing mRNA extracted from developing mouse embryos and a panel of adult tissues (CLON-TECH) were probed with a SIMPL cDNA radiolabeled by random priming [␣-32 P]dCTP (3000 mCi/ml) (Amersham Pharmacia Biotech) according to the manufacturer's recommendations.
Plasmid Constructs and Antibodies-The IL-8-Luc and (NF-B) 3 -Luc reporter constructs and IRAK/mPLK expression constructs have been described previously (24). The SIMPL cDNA was subcloned into a mammalian expression vector that placed the SIMPL coding region under the control of the cytomegalovirus immediate early gene promoter (pFLAG-CMV2, Eastman Kodak). The wild-type and catalytically inactive versions of IKK␣ and IKK␤ (15) were kindly provided by Michael Karin (University of California, San Diego). The mouse c-Myc monoclonal antibody 9E10 was purchased from Roche Molecular Biochemicals. Chromatographically purified mouse IgG was purchased from Zymed Laboratories, Inc. (South San Francisco, CA). IRAK/mPLK, IKK␣, and IKK␤ antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A peptide fragment corresponding to amino acids 200 -213 of SIMPL conjugated to keyhole limpet hemocyanin was used to immunize a female chicken (Gallus Immunotech, Ontario, Canada). Crude egg lysates were enriched for IgY-containing protein by affinity chromatography. In pilot studies, we determined that the SIMPL antiserum could be used for Western blot analysis but was not suitable for immunocomplexing assays (data not shown).
Cell Culture and Transfections-Human embryonic kidney 293 (HEK 293) epithelial cells and C3H10T1/2 mouse embryo fibroblast cell lines were maintained and transfected as described previously (24). To monitor transfection efficiencies, precipitates also included a construct containing the Renilla luciferase cDNA. Cultures were harvested 24 or 48 h following transfection, and luciferase activities were determined using the Dual-Luciferase ® reporter assay system (Promega, Madison, WI) according to the manufacturer's specifications. Individual assays were normalized for Renilla luciferase activity, and data are presented as the -fold increase in activity relative to empty vector control. Data are from 2 to 3 independent experiments performed in duplicate or triplicate with similar qualitative results with standard errors indicated.
TUNEL Assays-HEK 293 cells (2 ϫ 10 6 ) were transfected with a mammalian expression vector encoding ⌬SIMPL or ⌬SIMPL plus a mammalian expression vector encoding CrmA. To control for the presence of nonapoptotic cells, one set of cultures was transfected with empty vector. 24 h later cells were harvested, fixed, and permeabilized as described below. As a positive control, fixed permeabilized cells were treated with DNase I prior to analysis. To detect double strand DNA breaks, terminal deoxynucleotidyltransferase was used to add fluorescein-labeled nucleotides to free 3Ј breaks in the DNA strands with an in situ cell death detection kit (Roche Molecular Biochemicals). Flow cytometry was used to detect cells containing fluorescein-labeled DNA.
IRAK/mPLK Nulligenic ES Cells-IRAK/mPLK nulligenic ES cells were generated with the mPLK/IRAK-targeting construct as described by Thomas et al. (27). The 129 Sv-derived ES cell line R1 was maintained in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin, 2 mM glutamine, 1.0 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 1.2 ϫ 10 3 units/ml leukemia inhibitory factor (LIF, Life Technologies, Inc.) on monolayers of mitomycin C-treated STO cells (subline of SIM mouse fibroblasts). To introduce the targeting vector, 8 ϫ 10 6 R1-ES cells were electroporated in 0.8 ml of cold phosphate-buffered saline containing 40 g of the linearized targeting construct. Electroporated cells were incubated on ice for 20 min before co-culturing with the mitomycin-treated STO monolayers. Three days after electroporation, cell cultures were treated with 400 g/ml G418 (Life Technologies, Inc.) and 2 M gangcyclovir. Individual colonies were isolated 10 days later. Genomic DNA was purified from G418 and gangcyclovir-resistant ES cell colonies with a DNA isolation kit (Puregene, Minneapolis, MN). The first round of screening was performed by polymerase chain reaction, and positives were rescreened by Southern blot analysis. Genomic DNA was digested with the BamHI restriction endonuclease and separated on a 0.7% agarose gel in 1ϫ a buffer containing 40 mM Tris acetate and 1 mM EDTA. The DNA was transferred to a nylon membrane (MSI, Westboro, MA) and probed with genomic fragments that distinguish the wild-type and targeted allele using IRAK/mPLK genomic probes outside the region that is used to generate the targeting construct. Only one targeting event was necessary as the mPLK gene is located on the X chromosome.
Liquid Chromatography Electron-Spray Mass Spectroscopy-Immunocomplexed materials were subject to SDS-polyacrylamide gel electrophoresis, and individual protein bands were excised and digested with trypsin. Solubilized samples were analyzed by capillary liquid chromatography using an ABI 140D solvent delivery system. Samples were applied directly to 300-m inside diameter fused silica capillaries packed with Vydac C18 resin and separated with gradients of Buffer A (2% acetonitrile and 98% H 2 O containing 0.2% isopropyl alcohol, 0.1% acetic acid, and 0.001% trifluoroacetic acid). Peptides were eluted with a flow rate of 7 l/min directly into the electrospray ionization source of a Finnigan LCQ mass spectrometer. Nitrogen was used as the sheath gas with a pressure of 35 p.s.i., and no auxiliary gas was used. Electrospray ionization was conducted with a spray voltage of 4.8 kV, a capillary voltage of 26 V, and a capillary temperature of 200°C. Spectra were scanned over a range of 200 -2000 m/z. Base peak ions were trapped using a quadrupole ion trap and further analyzed with a high resolution zoom scan using an isolation width of 3 m/z and collisioninduced dissociation scans with a collision energy of 40.

RESULTS AND DISCUSSION
Identification of SIMPL-The mechanism through which IRAK/mPLK activates NF-B-dependent gene transcription is not completely understood. To identify regulators and/or substrates of IRAK/mPLK, the yeast two-hybrid system was used to screen for binding proteins (see "Experimental Procedures" for details). Because a Gal4-IRAK/mPLK fusion protein containing full-length IRAK/mPLK strongly activated all reporter genes alone, IRAK/mPLK deletion constructs were generated and tested for auto-activation. A Gal4-IRAK/mPLK fusion protein containing amino acids 1-533 of IRAK/mPLK (deleting amino acids 534 -710) was used for large scale two-hybrid analysis. Seven positive clones were identified and determined to contain an identical 1.05-kb insert encoding an open reading frame of 259 amino acids. The protein encoded by the clones identified in the two-hybrid screen is a denoted signaling molecule that associates with mPLK (SIMPL); the deduced amino acid sequence for SIMPL is shown in Fig. 1A. Data base searches identified expressed sequence tags for rat and human SIMPL homologues (Fig. 1A). Readily discernible protein motifs were not identified in SIMPL using several different algorithms, nor were Drosophila or Caenorhabditis elegans homologues.
The endogenous pattern of SIMPL gene expression was determined by Northern blot analysis of mRNA isolated from embryonic tissues and a panel of adult mouse tissues. A 1.3and 2-kb transcript hybridized to the radiolabeled SIMPL cDNA probe (Fig. 1, B and C). The SIMPL cDNA identified in our two-hybrid screen (1.05 kb) is most likely encoded by the 1.3-kb transcript. Whether the 2-kb transcript corresponds to a splice variant of SIMPL encoding a related protein or an unrelated cross-reacting mRNA has not been determined. SIMPL mRNA levels peak in the day 10 mouse embryo and steadily decline thereafter. In adult tissues, the highest levels of SIMPL expression is detected in the testis, brain, kidney, liver, and heart. Upon longer exposure SIMPL transcripts are detected in the lung and skeletal muscle.
SIMPL Interacts with IRAK/mPLK in Intact Cells-To determine whether an interaction between IRAK/mPLK and SIMPL can be detected in mammalian cells, immunocomplexing assays were performed. Antiserum to endogenous IRAK/ mPLK protein was used to generate immunocomplexes from HEK 293 cells. As a negative control, immunocomplexes were also generated with a nonspecific mouse IgG. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, and Western blots were prepared and probed with antiserum to IRAK/mPLK or antiserum to SIMPL. SIMPL protein is detected in immunocomplexes generated with the IRAK/mPLK antisera (Fig. 1D, lane under ␣IRAK). Neither IRAK/mPLK nor SIMPL is found in immunocomplexes generated with an unrelated mouse IgG (Fig. 1D, lane under IgG). Detection of IRAK/mPLK in immunocomplexes with SIMPL suggests that the interaction initially detected in the yeast two-hybrid screen can be detected in intact cells. In an independent experiment, a SIMPL-derived peptide was identified (Fig. 1A, underscored residues) in Myc-tagged IRAK/mPLK immunocomplexes isolated from fibroblasts that were subjected to liquid chromatography electron spraymass spectroscopy (see "Experimental Procedures" for details). Thus, the interaction between IRAK/mPLK and endogenous SIMPL can be detected using two independent analytical approaches.
Transactivation of NF-B-dependent Gene Expression by SIMPL-NF-B is a key regulator of the immune and stress responses in mammals, and NF-B activity is increased in response to a variety of stimuli (for review see Ref. 1). Thus, we examined whether SIMPL, like IRAK/mPLK, induces NF-B activity (24). In these experiments, two different reporter constructs were used: a luciferase cDNA under the control of NF-B-dependent IL-8 gene promoter (IL-8-Luc) and a luciferase cDNA under the control of three tandem NF-B sites ((NF-B) 3 -Luc). Transient transfection of a mouse embryo fibroblast cell line with a SIMPL cDNA results in a dose-dependent induction of IL-8-Luc and (NF-B) 3 -Luc activity (Fig. 2, A and B) with no effect on an activating protein-1-dependent reporter construct (data not presented). Therefore, like IRAK/mPLK, SIMPL lies in a signaling pathway upstream of NF-B.
With the goal of generating a SIMPL mutant that could be used to study the role of SIMPL in IRAK/mPLK-dependent signaling, a SIMPL mutant (⌬SIMPL), in which the first 80 amino acid residues are missing, was generated. In preliminary studies we noted that the expression of ⌬SIMPL decreased cell survival. 2 Thus, we examined directly whether ⌬SIMPL expression was pro-apoptotic. HEK cells were transiently transfected with an empty vector or ⌬SIMPL in the absence or presence of CrmA, a nonspecific caspase inhibitor from the cowpox virus (29). 24 h later, cultures were harvested and analyzed for the presence of DNA double strand breaks (in situ cell death detection kit, Roche Molecular Biochemicals). Fluorescence-activated cell sorting analysis revealed that the expression of ⌬SIMPL leads to an increase in the number of cells containing double-stranded DNA breaks as measured by an increase in fluorescence, which are not detected in the presence of CrmA (Fig. 2C). Based on these results, in subsequent experiments in which the ⌬SIMPL mutant was analyzed CrmA was also included. To determine whether the ⌬SIMPL mutant functioned as a dominant-negative, wild-type SIMPL was coexpressed with increasing amounts of ⌬SIMPL. Expression of ⌬SIMPL decreases in a dose-dependent manner, SIMPL-induced NF-B activity (Fig. 2D). Taken together, these results reveal that ⌬SIMPL functions as a dominantnegative allele of SIMPL, and SIMPL, like NF-B, appears to be critical for cell survival.
To define the functional relationship between IRAK/mPLK and SIMPL, we examined whether the ability of SIMPL to induce NF-B activity was IRAK/mPLK-dependent. For these studies, a clone of embryonic stem cells nulligenic for mPLK (ES Ϫ/Ϫ, see "Experimental Procedures") was analyzed and compared with wild-type ES cells (ES ϩ/ϩ). Overexpression of SIMPL in ES ϩ/ϩ cells increases IL-8-Luc promoter activity 3-fold (Fig. 3A). In contrast, overexpression of SIMPL in the ES Ϫ/Ϫ cells does not increase IL-8 promoter activity (Fig. 3A). Thus, the ability of SIMPL to induce NF-B activity is dependent upon the presence of the IRAK/mPLK protein. We next determined whether IRAK/mPLK-induced NF-B activation is SIMPL-dependent by examining whether ⌬SIMPL blocked IRAK/mPLK-induced NF-B activity. Coexpression of ⌬SIMPL with IRAK/mPLK inhibits IRAK/mPLK activity (Fig. 3B). Taken together, these data support the hypothesis that SIMPL is a component of an IRAK/mPLK-dependent pathway that controls NF-B activity.
Our group has identified a requirement for IRAK/mPLK catalytic activity in TNF-RI-dependent induction of NF-B ac- tivity that occurs in a TRADD-independent manner (24). Therefore, it was of great interest to determine whether SIMPL, like IRAK/mPLK, is required for TNF-RI-induced NF-B activity. Consistent with the hypothesis that SIMPL is downstream of IRAK/mPLK, ⌬SIMPL inhibits TNF-RI-induced NF-B activity (Fig. 3C). To determine whether ⌬SIMPL-induced inhibition is specific for TNF-RI, the effect of ⌬SIMPL on IL-1-induced NF-B activity was measured. Expression of ⌬SIMPL does not inhibit IL-1-induced NF-B activity (Fig. 3D). In parallel to the effect seen when catalytically inactive IRAK/mPLK is expressed (24 -26), ⌬SIMPL appears to enhance IL-1-dependent induction of NF-B activity. Thus, like IRAK/mPLK catalytic activity, SIMPL is also required for TNF-RI-dependent induction of NF-B activity.
IKK␣ and IKK␤ Mutants Inhibit IRAK/mPLK and SIMPLinduced NF-B Activation-Current models predict that IKK␣ and IKK␤ are required for the activation of NF-B-dependent gene expression (30 -33). Thus, we examined whether SIMPL-induced NF-B activation requires IKK␣ and/or IKK␤ activity. Substitution of the lysine residue at position 44 for methionine within IKK␣ (IKK␣KM) and for alanine within IKK␤ (IKK␤KA) results in the loss of IKK catalytic activity (15). IKK␣KM or IKK␤KA attenuate IRAK/ mPLK-induced NF-B activation and SIMPL-induced NF-B activation (Fig. 4, A and B, respectively). Thus, like TNF-RI (30 -33), IKK␣ and IKK␤ are necessary components of the pathway through which IRAK/mPLK and SIMPL signal for activation of NF-B. We next examined whether IKK-induced NF-B activity is affected by ⌬SIMPL expression. ⌬SIMPL inhibited IKK␣-and IKK␤-induced NF-B activity (Fig. 4C). These data suggest a model in which SIMPL integrates the activities of upstream activators, like IRAK/mPLK, with the IKK-containing complex(es).
To evaluate the role of the SIMPL protein in physical interactions between IRAK/mPLK and the IKKs, we first examined whether expression of a SIMPL antisense construct would decrease steady-state levels of SIMPL protein and protein activity. Mouse embryo fibroblasts were transfected with the IL-8 luciferase and Renilla luciferase reporter constructs, a mammalian expression vector encoding an epitope-tagged version of SIMPL (SIMPL-FLAG) and an increasing amount of a SIMPL antisense construct. Because the dominant-negative allele of SIMPL induced an apoptotic response, a mammalian expression vector encoding CrmA was included with the transfected DNAs. In the presence of the SIMPL antisense construct there is a dose-dependent decrease in SIMPL-induced NF-B activity (Fig. 5A) and a decrease in the steady-state levels of SIMPL protein (Fig. 5B). We also examined whether transfection with the SIMPL antisense construct would result in a decrease in the steady-state level of endogenous SIMPL protein. Consistent with the results obtained with the FLAG-tagged SIMPL construct, transfection of the SIMPL antisense construct leads to a decrease in the steady-state level of endogenous SIMPL protein (Fig. 5C). Thus, the transfection of HEK cells with the SIMPL antisense construct results in decreased SIMPL protein and protein activity.
The data presented thus far demonstrate that the SIMPL and IKK protein activities are interdependent. Consequently, we were interested in determining whether the IRAK/mPLK and SIMPL proteins could be found in IKK-containing complexes.
We first examined whether IRAK/mPLK-SIMPL could be found in IKK␣-and/or IKK␤-containing complexes. To test this hypothesis, IRAK/mPLK antiserum or a mouse IgG control was used to generate immunocomplexes that were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting. Analysis of the Western blot probed with the SIMPL antiserum revealed the presence of SIMPL in complexes obtained with the IRAK antiserum (Fig. 5D, middle lane) but not in complexes generated with a mouse IgG control (Fig. 5D, first lane). When the Western blot containing the immunocomplexes that were generated with the IRAK/mPLK antiserum was probed with antisera to IKK␣ or IKK␤, both proteins were detected (Fig. 5D, middle lane). Based on these data, we hypothesize that complexes containing IRAK/mPLK, SIMPL, IKK␣, and IKK␤ can be isolated from cells under steady-state conditions.
To determine whether there is a requirement for SIMPL in IRAK/mPLK⅐IKK␣/IKK␤ complex formation, we examined whether the SIMPL antisense construct would affect IRAK/ mPLK⅐IKK␣/IKK␤ complex formation. In these experiments the SIMPL antisense construct was introduced into HEK 293 cells, and immunocomplexes were generated with antibody to endogenous IRAK/mPLK protein. Western blots were prepared and probed with SIMPL, IRAK/mPLK, IKK␤, and IKK␣ antisera. In the IRAK/mPLK-containing immunocomplexes isolated from cultures expressing the antisense SIMPL construct, neither the SIMPL protein nor the IKK␤ proteins were detected in association with IRAK/mPLK (Fig. 5D, last lane). Intriguingly, the IKK␣ protein was detected in the IRAK/ mPLK protein complexes independent of SIMPL.
In summary, SIMPL is a novel component of the IRAK/mPLKdependent TNF-RI signaling pathway that leads to the activation of NF-B. Several different sets of data support a link between SIMPL and TNF-RI-dependent activation of NF-B. RelA Ϫ/Ϫ animals die of massive liver apoptosis on or about 15-16 days of embryogenesis (34), a defect that does not occur in RelA Ϫ/Ϫ TNF Ϫ/Ϫ animals (35). Consistent with these data, SIMPL transcripts are found in the liver. Overexpression of wild-type SIMPL leads to induction of the NF-B activity, which is associated with cell survival, and high levels of SIMPL transcripts are detected in the brain and testis, immune-privileged tissues in which cell survival is paramount. Recently, Pfeuffer et al. (36) isolated a truncated version of SIMPL (amino acids 52-259) in a two-hybrid screen for proteins that bind ActA, a critical factor in the pathogenesis of a Listeria monocytogenes infection. Intriguingly, unlike wild-type animals, a L. monocytogenes infection in TNF Ϫ/Ϫ or TNF-RI Ϫ/Ϫ animals is lethal (37,38).
Several groups of investigators including ourselves have demonstrated that mPLK/IRAK catalytic activity is not required for IL-1 induction of an NF-B-dependent response (24 -26). Our group has identified a requirement for mPLK/IRAK catalytic activity in TNF-RI-dependent induction of NF-B activity that occurs in a TRADD-independent manner (24). Because dominant-negative SIMPL blocks a TNF-RI but not an IL-1-RI response, our data support a model in which SIMPL is a component of the mPLK/IRAK-dependent signaling pathway that requires IRAK/mPLK catalytic activity and lies downstream of TNF-RI.
The precise function of SIMPL is unclear. SIMPL appears to facilitate and/or regulate complex formation between IRAK/ mPLK-and IKK-containing complexes. The evidence presented herein support the existence of a TNF-RI-dependent IRAK/ mPLK-SIMPL-IKK␤-dependent signaling pathway. Tojima et al. (39) recently reported that NF-B-activating kinase couples protein kinase C activity to IKK␣/␤-containing complexes. These data combined with the results presented herein and elsewhere (for review see Ref. 40) suggest that ligand-dependent NF-B activation events that occur coordinate with IB protein phosphorylation and degradation converge at the level of the IKKs. FIG. 5. IRAK/mPLK, SIMPL, and IKK complex formation. A and B, HEK 293 cells were cotransfected with an IL-8-Luc reporter construct, a construct encoding the Renilla luciferase cDNA, a mammalian expression vector encoding CrmA, and the indicated constructs. The antisense SIMPL (asSIMPL) construct contains the SIMPL cDNA in reverse orientation under the control of the cytomegalovirus immediate early gene promoter. 24 h later cell lysates were prepared. In A, luciferase activities were measured as described under "Experimental Procedures." Error bars are mean Ϯ S.D. of duplicate samples. In B, lysates were used to generate Western blots, which were probed with antibody to the SIMPL protein. C, HEK 293 cells were transfected with a mammalian expression vector encoding CrmA and either an empty mammalian expression vector (Ϫlane) or a mammalian expression vector encoding an antisense SIMPL construct (ϩlane). 24 h later, cell cultures were harvested, lysates were prepared, and Western blots were generated and probed with antibody to the SIMPL protein. D, HEK 293 cells were transfected with a mammalian expression vector encoding CrmA plus either an empty mammalian expression vector (Ϫlane under ␣IRAK) or a mammalian expression vector encoding an antisense SIMPL construct (ϩlane under ␣IRAK). 24 h later, HEK 293 cells were harvested, cell lysates were prepared, and immunocomplexing assays were performed with a mouse IgG control (lane under IgG) or an antibody to IRAK/mPLK (lanes under ␣IRAK). Western blots were prepared and probed with antibody to mPLK/IRAK, SIMPL, IKK␣, and IKK␤. Bold arrows indicate the location of detected proteins.