Figure 3
Effect of LXN on ubiquitous degradation of IκBα. (A) HIEC cells were seeded in 6-well plates, 48 h after transfection of Flag-LXN or control plasmid, the cells were lysed and the expression of IĸBα and Flag-LXN were determined by Western blot and quantitated by image J. (B) HIEC cells were seeded in 6-well plates, 72 h after transfection of siLXN or siCTL, the cells were lysed and the expression of IĸBα and LXN were determined by Western blot and quantitated by image J. (C,D), HIEC (C) and HCT116 (D) cells seeded in 6-well plates were transfected with pFlag-LXN plasmid. 48 h after transfection, cells were treated with CHX (100 µg/mL) for 0, 4, 6 and 12 h, cell lysates were harvested, and IκBα was determined by western blot. Quantitative analysis of IκBα was performed by image J. (E) HEK293T cells were co-transfected with His-IκBα, Flag-LXN and HA-Ub plasmids as indicated. For ubiquitination assay, the cells were incubated in the presence of 10 μM MG132 for 12 h before assay. 48 h after co-transfection, immunoprecipitation was performed with anti-His antibody, and HA-antibody was used to detect the ubiquitylation of IκBα. (F) HCT116 cells were transfected with Flag-LXN plasmid. 48 h after transfection, cells were treated with TNF-α (20 ng/mL) for 30 min, immunoprecipitation was performed with anti-IκBα antibody and then separated by 10% SDS-PAGE, followed by immunoblotting with antibody as indicated. G, H CT116 cells were transfected with siLXN or siCTL for 72 h, and then the cells were stimulated with TNF-α (20 ng/mL) for 30 min, immunoprecipitation was performed with anti-IκBα antibody, followed by immunoblotting with antibody as indicated. Data are representative of at least three independent experiments. The results shown are the mean ± SEM. A two-tailed unpaired t-test was used to compare experimental groups. *p < 0.05; **p < 0.01.
