Interaction of LC8 with IκBα and its inhibition of IκBα phosphorylation by IKK. A, association of LC8 with IκBα. HeLa cells transfected for 24 h with pFLAG-LC8 were lysed in phosphate-buffered saline containing 1 mm EDTA, 0.5% Nonidet P-40, 1 mm AEBSF, aprotinin (10 μg/ml), and leupeptin (10 μg/ml). The cell lysates were incubated with normal rabbit IgG or antibody to IκBα (α-IκBα) for 2 h at 4 °C, and the immune complexes were then precipitated with protein A-Sepharose. Whole cell lysate (WCL) and immunoprecipitates (IP) were subjected to immunoblot analysis with antibodies to FLAG or to IκBα (upper panel). Alternatively, lysates of nontransfected HeLa cells were similarly subjected to immunoprecipitation, and endogenous LC8 was detected with antibody to LC8 (lower panel). B, inhibition by LC8 of IκBα phosphorylation mediated by IKK in vitro. An in vitro kinase assay was performed with recombinant IκBα, with IKK immunoprecipitated from cells treated (or not) with TNFα, and in the absence or presence of LC8, as described under “Experimental Procedures”. The relative activities of IKK were determined from the radioactivity (³²P) associated with IκBα bands. The reaction mixtures were also subjected to immunoblot analysis with antibody to IKKβ.

Augmentation of TNFα-induced NF-κB activation by depletion of LC8. A, HeLa cells were transfected for 60 h with LC8 siRNA or a control RNA and were then incubated in the absence or presence of TNFα (20 ng/ml) for 6 h. The abundance of MnSOD, LC8, and β-actin (loading control) in cell lysates was determined by immunoblot analysis. The intensity of the bands corresponding to MnSOD was normalized by that of the corresponding β-actin bands. B, HeLa cells were transfected as in A and incubated in the absence or presence of TNFα (20 ng/ml) for 1 h, after which total RNA was extracted from the cells and subjected to reverse transcription and real time PCR analysis to quantify the relative amounts of IκBα, cyclooxygenase-2, and IL-8 mRNAs. The data were normalized by the corresponding amount of β-actin mRNA and are the means ± S.D. of values from three independent experiments. C, HeLa cells were transfected as in A, incubated in the absence or presence of TNFα (10 ng/ml) for 7 min, fixed with 4% paraformaldehyde, and subjected to immunofluorescence staining as in Fig. 2B. D and E, HeLa cells transfected with LC8 siRNA as in A were incubated with TNFα (20 ng/ml) for the indicated times, after which cell lysates were subjected to immunoblot analysis with antibodies to p-IκBα, IκBα, p-IKKα/β, IKKβ, LC8, and β-actin (D). The chemiluminescence signals for p-IκBα were quantified and normalized by those of β-actin (E).

Reversible formation of an intermolecular disulfide bond by LC8 on oxidation. A, HeLa cells in six-well plates were transfected for 24 h with 1 μg of pFLAG-LC8 and then incubated with the indicated concentrations of H₂O₂ for 5 min. Free sulfhydryl groups of cellular proteins were masked with AMS as described under “Experimental Procedures,” and the samples were then subjected to SDS-PAGE under nonreducing (left panel) or reducing (right panel) conditions followed by immunoblot analysis with antibody to FLAG. B and C, HeLa cells were transfected for 24 h with expression vectors for wild-type (WT) or cysteine mutant forms of LC8 tagged with HA at the NH₂ terminus and were then incubated in the presence of 1 mm H₂O₂ for 5 min (B). Alternatively, COS7 cells were transfected for 48 h with expression vectors for the cysteine mutants of LC8 tagged with FLAG at the COOH terminus and were then exposed to 1 mm H₂O₂ for 10 min (C). The samples were prepared and analyzed under nonreducing conditions as in A, with the exception that N-ethylmaleimide was used instead of AMS and that antibody to HA was used to detect HA-LC8. Ox and Re indicate the oxidized and reduced forms of LC8, respectively.

Effects of BHA and DPI on the phosphorylation and degradation of IκBα during NF-κB activation by TNFα. HeLa cells were incubated in the absence or presence of 100 μm BHA (A–C) or 10 μm DPI (D–F) for 30 min and were then stimulated with TNFα (20 ng/ml) in the continued absence or presence of the chemicals for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to p-IκBα, to IκBα, or to tubulin (loading control) (A and D). The chemiluminescence signals corresponding to p-IκBα (B and E) and to IκBα (C and F) were quantified, normalized by those of tubulin, and plotted.

Model for the redox regulation of TNFα-induced NF-κB activation through LC8. See text for details.

Inhibition of TNFα-induced NF-κB activation by LC8 overexpression. A, HeLa cells were transfected for 24 h with either pFLAG-CMV2 (2 μg) or the indicated amounts of pFLAG-LC8, with 0.25 μg of pNF-κB-Luc (NF-κB reporter plasmid), and with 0.25 μg of pRL-SV40 (internal control). The cells were incubated in the absence or presence of TNFα (20 ng/ml) for 6 h, after which the luciferase activities of cell lysates were measured with a dual luciferase assay system as described under “Experimental Procedures” (lower panel); the data are the means ± S.D. of values from three independent experiments. Cell lysates from a representative experiment were also subjected to immunoblot analysis with antibody to LC8 (upper panel). B, HeLa cells transfected with either pFLAG-CMV2 (Mock) or pFLAG-LC8 for 24 h were grown on coverslips, incubated in the absence or presence of TNFα (10 ng/ml) for 15 min, and fixed with 4% paraformaldehyde. They were then subjected to immunofluorescence staining with antibody to p65 as well as to staining of nuclei with 4′,6-diamidino-2-phenylindole. C, HeLa cells were transfected with 1 μg of expression vectors for IKKβ or p65 as well as with pNF-κB-Luc, pRL-SV40, and the indicated amounts of pFLAG-LC8. They were harvested after 36 h for assay of luciferase activities. The data are the means ± S.D. of values from three independent experiments. D, HeLa cells were transfected with pFLAG-CMV2 or pFLAG-LC8 for 36 h and then incubated in the absence or presence of TNFα (20 ng/ml) for 10 min. The IKK complex was immunoprecipitated from cell lysates and assayed for kinase activity in vitro with IκBα as substrate as in Fig. 1B.

Inhibition by LC8 of NF-κB activation induced by IL-1β or LPS. A, HeLa, HEK293, and Raw 264.7 cells were transfected for 24 h with pNF-κB-Luc and pRL-SV40 as well as with pFLAG-CMV2 (Mock) or pFLAG-LC8. HeLa cells were then stimulated with TNFα (10 ng/ml) for 12 h, HEK293 cells were incubated with IL-1β (4 ng/ml) for 8 h, and Raw 264.7 cells were exposed to LPS (100 ng/ml) for 12 h. The cells were then harvested for assay of luciferase activities. The data are the means ± S.D. of values from three independent experiments. B and C, HeLa (B) or HEK293 (C) cells were transfected with LC8 siRNA or a control RNA for 60 h and were then either incubated in the absence or presence of LPS (100 ng/ml) for 12 h (B) or of IL-1β (10 ng/ml) for 6 h (C). The cell lysates were then analyzed for MnSOD expression as in Fig. 3A.

Redox-dependent interaction of LC8 with IκBα. A, HeLa cells were deprived of serum for 6 h in Dulbecco's modified Eagle's medium lacking phenol red, loaded with 2.5 μm CM-H₂DCFDA for 17 min, and then incubated in the absence or presence of TNFα (10 ng/ml) for 3 min. 2′,7′-dichlorofluorescein (DCF) fluorescence was visualized with a confocal laser-scanning microscope (upper panels). Relative fluorescence intensity per cell was measured by averaging the values for five groups of cells in each image and is presented as the means ± S.D. (lower panel). B, HeLa cells were transfected with pFLAG-LC8 for 24 h and then treated with TNFα (100 ng/ml) or 1 mm H₂O₂ for 10 min. The redox status of LC8 was analyzed as in Fig. 5B. C, recombinant LC8 (50 μm) was incubated with 1 mm DTT or 1 mm H₂O₂ for 1 h in a solution containing 20 mm Tris-HCl (pH 8.0) and 1 mm EDTA, after which the remaining DTT or H₂O₂ was immediately removed by ultrafiltration. His₆-tagged IκBα (1 μg) was incubated overnight at 4 °C with 1 μg of DTT-treated LC8 (re-LC8) or H₂O₂-treated LC8 (ox-LC8) in 0.5 ml of phosphate-buffered saline containing 0.5% Nonidet P-40, 1 mm AEBSF, aprotinin (10 μg/ml), and leupeptin (10 μg/ml). Protein complexes were then collected by incubation of the reaction mixtures with nickel-chelating Sepharose for 30 min at 4 °C followed by centrifugation, and they were subjected to immunoblot analysis with antibodies to LC8 and to IκBα (upper panels). The redox status of LC8 (lower panel) was also analyzed as in B. D, HeLa cells were exposed to 50 μm MG132 for 1 h and were then incubated in the absence or presence of 1 mm H₂O₂ or TNFα (100 ng/ml) for 10 min. Whole cell lysates (WCL) or immunoprecipitates (IP) prepared with antibody to IκBα as in Fig. 1A were subjected to immunoblot analysis of LC8 and IκBα. E and F, HEK293 cells were incubated overnight in Dulbecco's modified Eagle's medium supplemented with 0.5% fetal bovine serum. HEK293 or Raw 264.7 cells were incubated in Dulbecco's modified Eagle's medium without phenol red in the absence or presence of IL-1β (10 ng/ml) or LPS (1 μg/ml) for 30 min, respectively. After stimulation, the cells were immediately incubated with 2.5 μm CM-H₂DCFDA for 5 min, and 2′,7′-dichlorofluorescein fluorescence was then visualized with a confocal laser-scanning microscope (E). The relative fluorescence intensity per cell (F) was measured as in A.

Regulation of the redox status of LC8 by TRP14. A, reduction of oxidized LC8 by TRP14 was assayed at 30 °C by monitoring A₃₄₀ in a 0.2-ml reaction mixture containing 50 mm HEPES-NaOH (pH 7.0), 1 mm EDTA, 0.2 mm NADPH, 100 nm TrxR1, 170 μm oxidized LC8, and 10 μm TRP14 or TRP14-C43S. Oxidized LC8 was prepared as in Fig. 6C. A reaction mixture lacking TrxR1 served as a control. B, reduction of oxidized LC8 by TRP14 was initiated by the addition of oxidized LC8 at a final concentration of 50 μm to a reaction mixture containing 50 mm HEPES-NaOH (pH 7.0), 1 mm EDTA, 0.2 mm NADPH, 100 nm TrxR1, and 5 μm TRP14 or TRP14-C43S. The reaction was terminated after incubation for 30 min at 30 °C. As a positive control, 1 mm DTT was used for the reduction of oxidized LC8 under the same conditions. The redox status of LC8 was analyzed as in Fig. 6C. Ox and Re indicate the oxidized and reduced forms of LC8, respectively.