Figure 2
From: Regulation of ER stress-induced autophagy by GSK3β-TIP60-ULK1 pathway
Acetylation of ULK1 by TIP60 under ER stress. (a) TM-induced acetylation of ULK1. Endogenous ULK1, which was immunoprecipitated from cell lysates that were harvested after 24 h TM (10 μg/ml) treatment, was blotted with an antibody against acetylated lysine. The same membrane was reprobed with anti-ULK1 antibody. The total cell lysates was blotted with anti-P-Atg13 (S318) antibody and anti-Atg13 antibody. Ratio of acetylated ULK1 to total ULK1 and phosphorylated Atg13 to total Atg13 were calculated. Data represent the mean±S.E.M. of three independent experiments (*P<0.05 (Student’s t-test)). (b) The effect of GSK3β inhibitor on TM-induced acetylation of ULK1. HeLa cells were treated as described in Figure 1b. The total and acetylated ULK1 levels were determined following immunoprecipitation as described in (a). Total cell lysates were blotted as indicated. Data represent the mean±S.E.M. of three independent experiments (**P<0.01; NS, not significant (two-way analysis of variance (ANOVA) followed by Tukey’s test)). (c) The effect of TIP60 knockdown on TM-induced ULK1 acetylation. HeLa cells transfected with control (NC) or TIP60 small interfering RNA (siRNA) for 48 h were treated with TM (10 μg/ml) for 24 h. The lysates were analyzed for total and acetylated ULK1. Total cell lysates were blotted as indicated. Data represent the mean±S.E.M. of three independent experiments (**P<0.01; NS, not significant (two-way ANOVA followed by Tukey’s test)). (d) The effect of modulating TIP60 on TM-induced ULK1 acetylation. HeLa cells transfected with Vector, wild type (WT) or S86A TIP60 were treated with TM (10 μg/ml) and analyzed for ULK1 acetylation after immunoprecipitation. Total cell lysates were blotted as indicated. Data represent the mean±S.E.M. of three independent experiments (*P<0.05; **P<0.01; NS, not significant (two-way ANOVA followed by Tukey’s test)). (e–h) HeLa cells were treated with TG (1 μM). The lysates were blotted as indicated. The detection and analysis of acetylated ULK1, phosphorylated Atg13 (S318) and phosphorylated TIP60 (S86) were carried out as described in (a–d) after 16 h of TG exposure. Data represent the mean±S.E.M. of three independent experiments. (e) *P<0.05 (Student’s t test); (f–h) *P<0.05; **P<0.01; NS, not significant (two-way ANOVA followed by Tukey’s test)
