SNCG stabilizes MKK3/6 and promotes TGF-β-induced p38MAPK phopshorylation. (A) HepG2 and HeLa cells were transfected with siControl or siSNCG. Twenty-four hours later, the cells were treated with 5 ng/ml TGF-β for another 48h, followed by western blot analysis of indicated proteins. (B) HepG2 cells were transfected with 50 nM of siControl or siSNCG for 24h, followed by qRT-PCR analysis of SNCG, MKK3 and MKK6 transcription. The levels of transcripts in siControl-transfected cells were set as 1. Values represent mean ± SD. (n = 3). ***, p <0.001, compared with siControl-transfected cells. (C) HepG2 lysates were subjected to immunoprecipitation with anti-SNCG, anti-MKK3/6 or normal IgG, followed by western blot analysis of MEK3/6 and SNCG. (D) HepG2 cells were transfected with siControl or siSNCG. 24h later, the cells were treated with protein translation inhibitor cycloheximide (CHX, 100 μg/mL) for indicated periods, followed by western blot analysis of MKK3/6 and SNCG. The relative levels of MKK3/6 were plotted. For both siControl- and siSNCG-transfected cells, the levels of MKK3/6 at 0 h were set as 100%. (E) HepG2 cells were transfected with the empty vector or SNCG expression plasmid, and treated with CHX for indicated periods, followed by western blot analysis of MKK3/MKK6 and SNCG. The relative levels of MKK3/6 were plotted. For both vector- and SNCG-transfected cells, the levels of MKK3/6 at 0 h were set as 100%.