Fig. 4

Phosphorylation of SQSTM1 T269/S272 inhibits the S403 phosphorylation during proteasome inhibition. A AD293 and Hela cells were treated with MG132 (2 μM), and Bafilomycin A1 (25 nM), alone or in combination for 14 h. The whole-cell lysates were subjected to western blot analysis with indicated antibodies. B Quantitative analysis of results in A. C HeLa cells were transfected with plasmids expressing FLAG-SQSTM1(T269A/S272A) or FLAG-SQSTM1(T269E/S272D) for 24 h, and then treated with MG132 (1 μM) and Bafilomycin A1 (25 nM) for 14 h. The cells were then fixed and analyzed by immunostaining with phospho-SQSTM1 (S403)-specific antibodies (Red) and anti-FLAG (Meganta) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. D Quantitative analysis of results in C, at least 25 cells from three independent experiments were scored for each group. E HeLa cells were transfected with plasmids expressing p38γ or p38δ mutants for 24 h, and then treated with MG132 (2 μM) for 14 h. The whole-cell lysates were subjected to western blot analysis with indicated antibodies. F Quantitative analysis of results in E. G SQSTM1 knockout AD293 cells stably expressing indicated constructs were treated with or without MG132 (2 μM) for 14 h. The whole-cell lysates were subjected to western blot analysis with indicated antibodies. H Quantitative analysis of results in G. I AD293 cells and A375 cells were treated with MG132 (2 μM), and Doramapimod (50 μM), alone or in combination for 14 h. The whole-cell lysates were subjected to western blot analysis with indicated antibodies. J, K Quantitative analysis of results in I. Data are mean ± SEM of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001, NS no significance