Fig. 1

SQSTM1 S403 phosphorylation does not affect the autophagic degradation of ubiquitinated proteins during proteasome inhibition. A–B Hela cells and AD293 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. Data are mean ± SEM of three independent experiments; **P < 0.01, ***P < 0.001, NS no significance. C Hela cells stably expressing GFP-LC3 were treated with MG132 (1 μM) and Bafilomycin A1 (25 nM), alone or in combination for 14 h. The cells were fixed, the images were captured with confocal microscope. Scale bar: 10 μm. D Quantitative analysis of results in C. About 25 cells from three independent experiments were scored for each group. Data are mean ± SEM; ***P ≤ 0.001. E 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. F-G Hela cells were treated with MG132 (2 μM) alone, or combined with BX-795 (5 μM)/CX-4945 (10 μM)/SBI-0206965 (10 μM) for 14 h. The whole-cell lysates were subjected to western blot analysis with indicated antibodies. Data are mean ± SEM of three independent experiments; **P < 0.01, ***P < 0.001, NS no significance. H 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