Fig. 3

Phosphorylation of SQSTM1 S403 promotes its autophagic sequestration. A HeLa cells stably expressing EGFP-LC3B were transfected with plasmids expressing FLAG-SQSTM1(S403A) or FLAG-SQSTM1(S403E) for 24 h, and then treated with MG132 (1 μM) for 14 h. The cells were then fixed and analyzed by immunostaining with anti-UB-k48 (red) and anti-FLAG (meganta) antibodies. Scale bar: 10 μm. B SQSTM1 knockout AD293 cells stably expressing FLAG-SQSTM1(S403E) were treated with 1 μM MG132 alone or combined with Wortmannin (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. C Quantitative analysis of results in B. 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