IκB Kinase, a Molecular Target for Inhibition by 4-Hydroxy-2-nonenal*

In unstimulated cells, transcription factor NF-κB is retained in the cytoplasm by interaction with the inhibitory protein, IκBα. Appropriate cellular stimuli inactivate IκBα by phosphorylation, ubiquination, and proteolytic degradation, which allows NF-κB to translocate to the nucleus and modulate gene expression. 4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, inhibits activation of NF-κB in the human colorectal carcinoma cell line (RKO) and human lung carcinoma cell line (H1299). Pretreatment of cells with HNE dose-dependently suppresses tetradecanoylphorbol acetate (TPA)/ionomycin (IM)-induced NF-κB DNA binding activity and transactivation of luciferase-based reporter constructs. HNE pretreatment has no affect on TPA/IM-induced AP-1 DNA binding activity. HNE inhibits TPA/IM-induced degradation of IκBα in both H1299 and Jurkat T cells. The accumulation of IκBα parallels the inhibition of its phosphorylation. At doses that inhibit IκBα degradation, HNE inhibits IκB kinase (IKK) activity by direct reaction with IKK. Covalent adducts of HNE to IKK are detected on Western blots using antibodies against IKK or HNE-protein conjugates. Addition of dithiothreitol prevents HNE modification of IKK. Thus, HNE is an endogenous inhibitor of NF-κB activation that acts by preventing IKK activation and subsequent IκBα degradation.


In unstimulated cells, transcription factor NF-B is retained in the cytoplasm by interaction with the inhibitory protein, IB␣. Appropriate cellular stimuli inactivate IB␣ by phosphorylation, ubiquination, and proteolytic degradation, which allows NF-B to translocate to the nucleus and modulate gene expression. 4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, inhibits activation of NF-B in the human colorectal carcinoma cell line (RKO) and human lung carcinoma cell line (H1299). Pretreatment of cells with HNE dosedependently suppresses tetradecanoylphorbol acetate (TPA)/ionomycin (IM)-induced NF-B DNA binding activity and transactivation of luciferase-based reporter constructs. HNE pretreatment has no affect on TPA/IMinduced AP-1 DNA binding activity. HNE inhibits TPA/ IM-induced degradation of IB␣ in both H1299 and Jurkat T cells. The accumulation of IB␣ parallels the inhibition of its phosphorylation. At doses that inhibit IB␣ degradation, HNE inhibits IB kinase (IKK) activity by direct reaction with IKK. Covalent adducts of HNE to IKK are detected on Western blots using antibodies against IKK or HNE-protein conjugates. Addition of dithiothreitol prevents HNE modification of IKK. Thus, HNE is an endogenous inhibitor of NF-B activation that acts by preventing IKK activation and subsequent IB␣ degradation.
Aldehydes are products and propagators of oxidative stress (1). They are reactive electrophiles that form adducts to protein and DNA that have been detected in tissues from healthy human beings and individuals with various diseases (2)(3)(4)(5)(6). Consequently, aldehydes modulate the activities of numerous proteins, induce mutations, and alter cell cycle progression (7)(8)(9)(10)(11)(12). For example, malondialdehyde, a major carbonyl product of lipid peroxidation, is mutagenic and carcinogenic and induces cell cycle arrest at the G 1 /S and G 2 /M checkpoints (7). The G 1 /S arrest in human colon and lung cancer cells (RKO and H1299, respectively) is caused by induction of the cyclin-dependent kinase inhibitor, p21, whereas the G 2 /M arrest appears to be due to a reduction in the level of the cdc2 kinase. Thus, alteration of gene expression triggered by protein or DNA damage may contribute to the range of biological effects exerted by aldehydes.
A panoply of pathophysiological responses is also exerted by 4-hydroxynonenal (HNE), 1 the major toxic product of lipid peroxidation (1). HNE reacts with sulfhydryl and amino groups and leads to inactivation of DNA polymerases, dehydrogenases, and various transporters, inter alia (13). It also causes cell cycle arrest and apoptosis (8 -10). HNE treatment of cells alters the expression of several transcription factors including c-Myc (12), c-Myb (14), and c-Jun (15), suggesting that it may have more global effects on protein expression and cell function. The induction of c-Jun by HNE is associated with activation of JNK kinase and p38 kinase, perhaps by H 2 O 2 modulation of upstream signaling pathways (15,16). A major signaling pathway associated with inflammation and oxidative stress is mediated by the transcription factor NF-B (17)(18)(19). NF-B consists of heterodimers of two polypeptides, p50 and p65, which are members of a family of proteins related to the proto-oncogene c-rel (20,21). Inactive NF-B is located in the cytosol, bound to its inhibitory protein, IB. Dissociation of NF-B from IB is a critical step in NF-B activation that leads to translocation of NF-B to the nucleus, enabling DNA binding and transactivation (22). This process is triggered by sequential phosphorylation and ubiquitination of IB␣, followed by digestion of the ubiquinated protein by the proteasome (23)(24)(25). The enzyme that catalyzes the ubiquitination of phosphorylated IB is constitutively active. Hence, in most cases, the key event for NF-B activation is phosphorylation of two serine residues at the N terminus of IB by IB kinase (IKK) (23,24).
We report here that treatment of RKO and H1299 cells with HNE leads to a dramatic loss of DNA binding and transcriptional activation by NF-B in cells treated with tetradecanoylphorbol acetate (TPA) and ionomycin (IM). The loss of NF-B activity is due to stabilization to the IB␣-NF-B complex, which results from a decrease in the rate of turnover of IB␣. The prevention of IB␣ turnover is attributable to the inhibition of IKK caused by direct reaction with HNE. These findings indicate that HNE is a potent inhibitor of the NF-B-dependent cell signaling.

EXPERIMENTAL PROCEDURES
Cell Lines, Culture Conditions, and Chemical Treatment-Human colorectal carcinoma cells (RKO) were maintained in McCoy's 5A medium (Hyclone, Logan, Utah). Human large cell lung carcinoma cells (H1299) were maintained in F-12 medium (Hyclone), and human lymphoma Jurkat T cells were maintained in RPMI (Hyclone). RKO and H1299 cells were grown in the presence of 10% bovine serum, and Jurkat T cells were grown in the presence of 10% heat-inactivated fetal bovine serum. All media were supplemented with 100 units/ml penicillin and 100 g/ml streptomycin. Cells were maintained in 5% CO 2 at 37°C. RKO and H1299 cells were plated 18 -24 h prior to chemical exposure and were 50 -70% confluent at a density of 7 ϫ 10 5 /ml at the time of treatment. HNE (a generous gift from V. Amarnath, Vanderbilt University) and TPA (Sigma) were dissolved in 70% ethanol, and IM (Calbiochem, San Diego, CA) was dissolved in Me 2 SO. The final concentration of ethanol or Me 2 SO in the medium was Յ0.1%.
To assess NF-B binding, nuclear extracts (5-10 g) were incubated with the labeled DNA probe in the presence of poly(dI-dC) (2 g), bovine serum albumin (2 g), and Nonidet P-40 (0.5%) in 20 l of reaction buffer (10 mM HEPES (pH 7.9), 20 mM KCl, 0.5 mM EDTA, 2.5 mM DTT, and 4% Ficoll) at room temperature for 10 min. Similarly, AP-1 binding was evaluated by incubating nuclear extracts (5-10 g) in reaction buffer with 1.5 mM MgCl 2 and 5 mM DTT in the presence of labeled DNA probe, poly(dI-dC) (2 g), and bovine serum albumin (2 g) at room temperature for 10 min. The specificity of binding was examined both by competition with unlabeled oligonucleotide and by supershift assay with respective antibodies. In supershift experiments, antibodies (1-2 g) directed against NF-B/p65, NF-B/p50, or AP1/c-Jun (Santa Cruz Biotechnology) were incubated with nuclear extracts for 45 min at 4°C before addition of labeled probe. In competition experiments, antibodies were replaced with a 50-fold molar excess of unlabeled oligonucleotide.
Binding activity was analyzed by electrophoretic mobility shift assay using a 4 -5% polyacrylamide gel and Tris, glycine, EDTA buffer (5 mM Tris (pH 8.4), 9 mM glycine, and 0.2 mM EDTA). Visualization and quantification of radioactive bands were performed using a Phospho-rImager (Molecular Dynamics).
Luciferase Assay-Cells were plated in 6-well plates at a density of 4 ϫ 10 5 cells per well 18 -24 h prior to transfection. Cells were transfected with 0.5 g of reporter construct in a pGL2 luciferase-expressing vector (Promega) using LipofectAMINE reagent (Life Technologies, Inc.). The plasmid was constructed with 6ϫ NF-B binding sites upstream of the SV40 promoter. Thus, the luciferase reporter gene was under NF-B control. Cells were exposed 18 -24 h after transfection to TPA/IM (0.08 M/2 M) or TPA/IM plus HNE (10 -80 M) for 6 h and lysed in luciferase reporter lysis buffer (Promega). Total cell lysates (5-10 g of protein) were determined for luciferase activity by the luciferase assay reagent (Promega) in a Monolight 2010 luminometer (Analytical Luminescence Laboratory).
Kinase Assay-To determine IKK activity, total cell extracts (200 -250 g of protein) were rotated with anti-IKK␣ antibody (2 g/ml) in KLB containing 0.5 M NaCl for 1 h at 4°C and then for an additional 2 h with protein A-Sepharose beads (Amersham Pharmacia Biotech). The immunoprecipitates were washed twice with KLB, 0.5 M NaCl and twice with kinase buffer (20 mM HEPES (pH 7.5), 10 mM MgCl 2 , 0.1 mM Na 3 VO 4 , 10 mM ␤-glycerophosphate, 1 mM DTT, 50 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, and protease inhibitors). The washed beads were incubated with 20 l of kinase buffer containing 2 g of GST-IB␣ substrate, 50 M ATP, and 2 Ci of [␥-32 P]ATP for 30 min at 30°C. The reactions were stopped by addition of 5ϫ SDS-PAGE sample buffer. The proteins were resolved by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. IKK activity was evaluated from the formation of ␥-32 P-IB␣-GST by autoradiography. The levels of IKK␣ and GST-IB␣ were analyzed by Western blot and ink staining, respectively.
HNE Inhibition of IKK Activity-Total cell extracts (200 -250 g of protein) were immunoprecipitated with anti-IKK␣ antibody and bound to protein A-Sepharose as described above. Immunoprecipitates served as the enzyme source for testing the effect of HNE on IKK activity in vitro. Prior to use, immunoprecipitates were washed twice with KLB, 0.5 M NaCl and twice with kinase buffer with or without DTT (1 mM). Cleared and washed beads were treated with HNE in 30 l of kinase buffer in the presence or absence of DTT (1 mM) for 10 min at 30°C. HNE treatment was terminated by adding 300 l of ice-cold kinase buffer with 1 mM DTT. Following treatment, beads were centrifuged and subjected to kinase assay as described.

HNE Blocks DNA Binding Activity of NF-B but Not AP-1/ c-Jun-
To determine whether HNE initiates a cellular response that suppresses NF-B DNA binding, we evaluated the effects of HNE treatment on NF-B activity in H1299 cells stimulated with TPA and IM. H1299 cells are derived from a large cell lung carcinoma and are p53-deficient. Fig. 1 demonstrates that treatment of H1299 cells with TPA/IM resulted in enhanced NF-B DNA binding activity in nuclear extracts. Increased activity was detectable within 5 min, reached a maximum at 15 min, and declined after 30 min of TPA/IM stimulation. The specificity of the nuclear binding activity was confirmed by competition of the labeled probe with a 50-fold excess of unlabeled NF-B oligonucleotide (Fig. 1A, lane 9). No competition was observed with an excess of an oligonucleotide containing an SP-1 sequence (data not shown). Treatment of the extracts with either anti-NF-B p50 or anti-NF-B p65/Rel A antibodies yielded supershifted bands (Fig. 1A, lanes 10 and  11). Hence, the binding is indeed NF-B-specific. Pretreatment of H1299 cells with 40 M HNE for 30 min completely prevented the TPA/IM-dependent increase in NF-B DNA binding activity. In fact, HNE pretreatment lowered the basal levels of NF-B binding activity 2-fold (Fig. 1A, compare lanes 1 and 5).
The inhibitory effect of HNE was not restricted to H1299 cells. A similar inhibition of TPA/IM-stimulated increase in NF-B binding was observed in RKO cells. RKO is a human colon carcinoma that is wild-type for p53 but mismatch repairdeficient. The effects of HNE in RKO cells were not as dramatic as in H1299 cells, because of the higher basal level of activity in the former (data not shown).
To determine whether the inhibitory effect of HNE was a nonspecific effect, we performed similar experiments probing for an alteration in TPA/IM-stimulated AP-1/c-Jun DNA binding activity. Using the same preparations of nuclear protein from H1299 cells that were used for the experiment summarized in the legend to Fig. 1A, we found that AP-1 binding activity was neither stimulated by TPA/IM nor inhibited by HNE (Fig. 1B). Binding specificity was confirmed, as before, by competition with unlabeled probe and supershift with anti-AP-1 antibody (Fig. 1B, lanes 9 and 10, respectively). The supershift in this particular experiment was relatively weak. Clearly, concentrations of HNE that completely prevent DNA binding by NF-B have no effect on DNA binding by AP-1.
HNE Blocks NF-B Transactivation in H1299 Cells-To correlate HNE effects on NF-B transactivation with DNA binding inhibition, an NF-B-dependent, luciferase-expressing vector was employed. Twenty-two h after transient transfection with the luciferase reporter, H1299 cells were stimulated with TPA/IM or treated with HNE and TPA/IM for 6 h. Control cells were not treated with HNE or with TPA/IM. Cell extracts were prepared and analyzed for luciferase activity. TPA/IM treatment induced a 3-fold increase in luciferase activity relative to untreated cells (Fig. 2). HNE treatment suppressed the TPA/ IM-induced increase in luciferase activity in a dose-dependent manner, with 20 M HNE providing complete suppression and higher doses decreasing luciferase activity to below unstimulated levels (Fig. 2). Thus, HNE inhibited both NF-B DNA binding and NF-B transcriptional activation. Parallel experiments with RKO cells produced similar results, as shown in the lower panel of Fig. 2.
HNE Blocks IB␣ Degradation in H1299 Cells and Jurkat T Cells-NF-B activation requires degradation of the inhibitory protein, IB␣ (26,27). Consequently, HNE inhibition of NF-B DNA binding and transactivation activities could result from the inhibition of IB␣ degradation. To test this possibility, the effects of TPA/IM stimulation and HNE treatment on IB␣ degradation were evaluated. Treatment of H1299 or Jurkat T cells with TPA/IM for 0 -30 min at 37°C resulted in a rapid decrease in cellular IB␣ protein (Fig. 3). For H1299 cells, the reduction in the level of IB␣ protein appeared maximal by 5 min, and some increase was evident by 30 min (Fig. 3A, lanes  1-4). For Jurkat T cells, the reduction in the level of IB␣ protein was detectable at 5 min, with complete disappearance evident in 20 min (Fig. 3B, lanes 1-4). In Jurkat cells, the TPA/IM-mediated decrease in cellular IB␣ concentrations resulted from an induction of IB␣ phosphorylation followed by a degradation of phosphorylated IB␣ (p-IB␣, Fig. 3B, lanes  1-4). In contrast, no detectable p-IB␣ was found in TPA/IMtreated H1299 cells (Fig. 3A, lanes 1-4). Pretreatment of cells with HNE prevented the TPA/IM-mediated reduction of IB␣ concentration (Fig. 3, A and B, lanes 5-8). In addition, HNE pretreatment completely abolished the formation of p-IB␣ in Jurkat T cells. Thus, it appears likely that HNE treatment prevents IB␣ degradation by inhibition of IB␣ phosphorylation.
HNE Inhibits IKK Activity in Jurkat T Cells-IKK activity is required for IB␣ phosphorylation (28 -30). Thus, one possible mechanism to explain the inhibitory effect of HNE on TPA/IM stimulation of NF-B activity is that HNE inhibits IKK activity. To test this possibility, Jurkat T cells, with or without a 30-min pretreatment with HNE, were stimulated with TPA/ IM. Total cell extracts were prepared, and IKK activity was determined using a fusion protein of IB␣ and glutathione S-transferase (IB␣-GST) as substrate. Kinase activity was evaluated by incorporation of 32 P into the fusion protein substrate. Incubation of the IKK substrate with TPA/IM-stimulated cell extracts resulted in a time-dependent increase in IB␣ phosphorylation (Fig. 4, lanes 1-4). Pretreatment of cells with HNE significantly inhibited the formation of 32 P-labeled IB␣-GST. This suggests that HNE inhibition of NF-B activation is due to inhibition of IKK activity.
HNE Blocks IKK Activity in Vitro-To clarify whether HNE inhibition of IKK activity occurs by direct interaction with IKK, an in vitro assay for HNE-mediated inhibition of IKK activity was developed. Immune complexes of IKK were precipitated, then incubated with HNE in the presence or absence of DTT for 10 min at 30°C, and assayed for IKK activity, as shown in Fig.  5. Treatment of immune complexes of IKK with HNE in the absence of DTT caused dose-dependent inhibition of IKK activity (Fig. 5A). Addition of 30 M HNE resulted in clear inhibition of IKK activity, and 60 M HNE completely inhibited activity. In fact, the higher dose of HNE lowered IKK activity to below basal levels. When parallel incubations were conducted in which immune complexes were treated with HNE in the presence of an excess of the HNE scavenging agent DTT (1 mM), only a modest decline in IKK activity was detected at the higher HNE concentration.
Western blots were performed to probe for the modification of IKK protein by HNE. Incubations of immune complexes of IKK with HNE produced bands that migrated more slowly than IKK as well as bands that migrated at the anticipated size for a dimer of IKK subunits (ϳ220 kDa) (Fig. 5A, lower two panels,  lanes 4 -5). Thus, incubation mixtures of IKK with HNE contained HNE-modified IKK molecules, some of which migrated as cross-linked protein dimers. Comparison of the kinase assay bands in Fig. 5A with the Western blots in the lower panels indicates that the formation of the HNE⅐IKK conjugates correlated with the loss of IKK activity. When parallel incubations of immune complexes of IKK and HNE were conducted in the presence of an excess of DTT, only trace amounts of slower migrating forms of IKK were detected; no higher molecular size HNE⅐IKK complexes were evident on gel electrophoresis (Fig.  5B, lower two panels, lanes 4 -5). These results demonstrate that HNE reacts covalently with IKK, which prevents IB␣ degradation and NFB activation. DISCUSSION In the present studies, we show that TPA/IM stimulates IB␣ phosphorylation and subsequent degradation, resulting in NF-B activation. This finding is consistent with previous observations that NF-B activation is responsive to a wide range of activators that lead to phosphorylation and degradation of IB␣ (19,26,27,31,32). Our experiments demonstrate that pretreatment of human cancer cells or Jurkat T cells with HNE leads to the inhibition of the NF-B signaling pathway. HNE prevents IB␣ phosphorylation and subsequent degradation, reducing NF-B DNA binding activity and NF-B transactivation. These results are in good agreement with the findings that HNE modulates NF-B activation by inhibiting IB␣ phosphorylation and subsequent proteolysis in human monocytic cells (33).
Interestingly, the complete process of IB␣ phosphorylation and subsequent degradation following treatment of cells with TPA/IM was only observed in Jurkat T cells. Phosphorylation of IB␣ was not observed in H1299 cells even though its TPA/ IM-stimulated degradation was obvious (Fig. 3). Three possibilities may explain the inability to detect p-IB␣ in H1299 cells. TPA/IM-induced phosphorylation of IB␣ may occur at a residue other than Ser-32 or Ser-36, so that the phosphorylated protein may not be recognized by the antibody employed in these studies. This possibility has been documented with anoxia, which stimulates phosphorylation of IB␣ at Tyr-42 and NF-B activation without proteasome-mediated degradation of IB␣ (34). A second possibility is that activation of NF-B in H1299 cells results from phosphorylation-independent IB␣ degradation. For example, UV irradiation leads to IB␣ degradation without phosphorylation in HeLa cells, 293 cells, and human fibroblasts (35,36). Finally, the kinetics of IB␣ phosphorylation and p-IB␣ degradation in H1299 cells may prevent a detectable steady-state concentration of p-IB␣ from accumulating. The kinase activity associated with the immunoprecipitate was determined using IB␣-GST fusion protein as a substrate. Equal amounts of the IB␣-GST substrate and the immunoprecipitated kinase complex were present in the assay, as confirmed by ink staining and immunoblotting of the membrane for the IB␣-GST and the IKK␣, respectively. Some random variation in the levels of IKK␣ was observed in individual experiments (e.g. lanes 6 and 7). The results are representative of two independent experiments.
Phosphorylation of IB requires IKK activity (22). IKK is a complex, which contains two catalytic subunits, IKK␣ (IKK1) and IKK␤ (IKK2), along with a regulatory protein, IKK␥ (37)(38)(39)(40). In our experimental conditions, both IKK␣ and IKK␤ were immunoprecipitated by anti-IKK␣ antibody (data not shown). Thus, the IKK activity represented the combination of IKK␣ and IKK␤. A variety of stimuli modulate the signal transduction pathways that lead to activation of upstream kinases including NF-B-inducing kinase and mitogen-activated protein kinase kinase kinase 1. These kinases are responsible for phosphorylation and activation of IKK (29,30,41). HNE did not inhibit any of these upstream kinases, and in fact, a brief survey indicated that it stimulated the activity of ERK1, ERK2, JNK1, and JNK2 (data not shown). This is consistent with a previous finding of stimulation of p38 kinase activity by HNE (42).
The effects of HNE are directly on IKK activity and appear to result from covalent modification of IKK protein. Aspirin, salicylate, and sulindac inhibit IKK␤ activity by competing for binding to ATP (43,44), whereas anti-inflammatory cyclopentenone prostaglandins inhibit NF-B activation by covalently modifying Cys-179 on the activation loop of IKK␤, leading to substantially reduced IKK␤ activity (45). It is well known that HNE can rapidly react with proteins containing sulfhydryl groups by Michael addition; so it is possible that HNE inhibits IKK activity by direct reaction with a cysteine residue (16,46). To test this possibility, we conducted an in vitro assay to assess the effect of HNE on IKK activity and protein modification. Our results demonstrated that HNE induced the loss of IKK activity concomitant with the formation of higher molecular size forms of IKK (Fig. 5A). A prominent band was detected at a molecular size corresponding to cross-linked homodimers or heterodimers of IKK subunits. The higher molecular size band on SDS-PAGE gels reacted with antibodies specific for IKK␣ and with antibodies specific for a Michael addition product of HNE with protein residues. This is consistent with the formation of an HNE-mediated cross-link of IKK protein subunits. The activation domains of IKK␣ or IKK␤ are believed to be located in close proximity to each other in the IKK complex, which might place the cysteine residues of the two activation domains close enough to enable cross-link formation (22,45).
The importance of the reaction of HNE with cysteine residues is suggested by the observation that treatment with DTT inhibited HNE-induced cross-link formation and loss of enzyme activity. DTT is a dithiol that is used as a reducing agent to protect free protein thiols from oxidation; it is commonly added to enzyme assays or purification buffers for this purpose. DTT also reacts with ␣,␤-unsaturated carbonyl compounds such as HNE. Its inclusion in the present experiments abolished modification and inhibition of IKK in cell-free extracts. We believe this accounts for previous reports that HNE does not inhibit IKK activity (33).
We demonstrate here that the key target in HNE modification of NF-B activity is IKK. Inhibition of IKK activity by this major product of lipid peroxidation occurs through covalent modification of the constituent proteins. Because NF-B stimulates transcription in response to oxidative stress, HNE modification may limit the magnitude of this transcriptional response. A similar role was recently proposed for 15-deoxyprostaglandin J 2 , which is a decomposition product of prostaglandin D 2 , a product of arachidonic acid metabolism in inflammatory cells (45,47). Furthermore, a related reaction with IKK may account for the previously noted inhibition of NF-B by acrolein (48). HNE is structurally related to 15-deoxyprostaglandin J 2 and acrolein, because it contains an ␣,␤-unsaturated carbonyl compound capable of reacting as a bifunctional electrophile. In this way, it may serve as an endogenous factor that  A and B). Individual samples were divided in two, and separate PAGE gels were run for Western blotting. After Western transfer, the blots were visualized with antiserum to IKK␣ or to HNE-modified protein. The third panel of A and B represents the detection of IKK molecules with an antiserum against IKK␣. The lower panel of A and B represents the detection of HNE-modified IKK molecules with an antiserum that recognizes HNE-modified protein conjugates. The amounts of IKK immune complexes added to each reaction corresponded to equal amounts of cell lysate. These complexes contained comparable amounts of IKK protein, as judged by the Western blots in A and B, lanes 1-3. Incubation with HNE may alter immune reactivity; so the amounts of IKK detected in A and B, lanes 4 -5, may not accurately reflect IKK content. The results are representative of three independent experiments. regulates the inflammatory response associated with oxidative stress.