Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates (original) (raw)
Fig 2
Phosphorylation of p62 at S409 is enhanced upon proteasome inhibition.
A. ULK1 mediates p-S409 of p62 upon MG132 treatment. HEK 293T cells transfected with empty vector, Myc-ULK1 WT or KI were treated with MG132 and then cell lysates were analysed with indicated antibodies(Top). The ratio of p-p62 and p62 was obtained by dividing the level of p-p62 by total p62(n = 3). One sample _t_-test was used and data are represented as mean ± SEM(n = 3). * p < 0.05; ns, not significant(bottom). B. ULK1 is important for MG132-induced p-S409 of p62. WT and ULK1 KO MEFs were treated with MG132 and analyzed with indicated antibodies(Top). The ratio of p-p62 and p62 was obtained by dividing the level of p-p62 by total p62(n = 3). One sample _t_-test was used and data are represented as mean ± SEM(n = 3). * p < 0.05; ns, not significant(bottom). C. MG132 treatment but not starvation increases p-S409 levels of p62 or ULK1-p62 interaction. p62 KO MEFs stably expressing empty vector or FLAG-p62 were incubated with MG132 or starved for glucose(-glu) or amino acid(-aa). F.M. indicates full medium as a control. Subsequently, IP with anti-FLAG or-ULK1 antibodies were performed and immunoprecipitants were analyzed with indicated antibodies.