Fig. 2

Phosphorylation of SQSTM1 S403 suppresses the aggresome formation of ubiquitinated proteins during proteasome inhibition. A Hela cells were treated with MG132 (1 μM) alone or combined with CX-4945 (5 μM) for 14 h. The aggresome formation was analyzed by immunostaining with anti-UB-K48 antibodies. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. B Quantitative analysis of results in (A). C HeLa cells were transfected with plasmids expressing FLAG-SQSTM1 wildtype (WT), FLAG-SQSTM1 (403A), FLAG-SQSTM1 (403E) for 24 h, and then treated with 1 μM MG132 for 14 h. The aggresome formation was analyzed by immunostaining with anti-UB-K48 (Red) and anti-FLAG (Green) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. D Quantitative analysis of results in C. E SQSTM1 knockout AD293 cells stably expressing indicated constructs were treated with 1 μM MG132 for 14 h. The aggresome formation was analyzed by immunostaining with anti-UB-K48 (Red) and anti-FLAG (Green) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. F Quantitative analysis of results in E. For B, D, and F, at least 50 cells were randomly selected from each group to score for aggresomes. Data are mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01