Fig. 5

CMTM3 reduces NEMO degradation. a The immunoprecipitation analysis of GES-1 cells. Cells expressing pCMV-NEMO-3Flag were immunoprecipitated by an anti-Flag antibody and immunoblotted by anti-NEMO and anti-CMTM3 antibodies. b Co-localization of CMTM3 and NEMO in GES-1 cells was investigated by immunofluorescence co-localization analysis after H. pylori infection for 24 h. Scale bar, 10 μm. Hochest was used for nuclear staining. c, d CMTM3 knockout GES-1 cells were treated with cycloheximide (25 µg/mL) and proteins were subjected to immunoblotting (c). Data is representative of three independent experiments. The remaining NEMO was quantified (d). e The ubiquitylation of NEMO. HEK 293 T cells were transfected with the indicated vectors. After 48 h transfection, cells were treated with MG132 (10 μM) for 6 h. Then cells were lysed and immunoprecipitated with anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. f CMTM3 KO GES-1 cells were treated with MG132 (10 μM) and collected at indicated time points. Whole-cell lysates were subjected to immunoblotting with indicated antibodies. g The expression the phosphorylated IKBα (p-IKBα) and phosphorylated p65 (p-p65) were analyzed in CMTM3 KO GES-1 cells.*p < 0.05