ULK1 mRNA and protein levels are regulated in a controlled time course during autophagy. (A) HeLa cells were treated with EBSS for the indicated time periods in the presence or not of Clq, and the levels of ULK1, ACTIN, and LC3 were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (right graph). Data are expressed as the mean ± SEM (n = 4). (B) qPCR analysis of ULK1 mRNA levels in HeLa cells incubated in EBSS medium for the indicated time periods. Data are expressed as mean ± SEM (n = 3). (C) qPCR analysis of ULK1 mRNA levels in HeLa cells incubated in EBSS medium for 4 h in the presence or not of Act D. Data are expressed as mean ± SEM (n = 3). (D) HeLa cells were treated with EBSS for the indicated time periods in the presence or not of MG132. Protein levels of ULK1 and TUBULIN were detected by WB. (E) HeLa cells were transfected with ubiquitin-HA and treated with EBSS for the indicated time periods in the presence of MG132. Protein extracts were immunoprecipitated in denaturing conditions using an anti-ULK1 antibody; ubiquitin, ULK1, and ACTIN were analyzed by WB. In A and B, data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. In C, data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison post test. ***, P < 0.001.