Figure 4. NEDD4L degrades ULK1 during prolonged starvation. (A) NEDD4L expression was down-regulated in HeLa cells using specific RNAi oligonucleotides (oligos; siRNA NEDD4L#1) or unrelated oligos as negative control (siRNA CTRL). Densitometric analysis of ULK1 over ACTIN is also shown (right graph). Data are expressed as the mean value ± SD (n = 3), and statistical analysis was performed using an unpaired Student's t test. **, P < 0.01. (B) NEDD4L expression was down-regulated in HeLa cells using specific RNAi oligos targeting NEDD4L 3′UTR (siRNA NEDD4L#2) or unrelated oligos as negative control (siRNA CTRL). Some of them were then transfected with empty vector or NEDD4L-HA. Levels of ULK1, NEDD4L, and ACTIN were detected by WB. Densitometric analyses of both ULK1 and NEDD4L over ACTIN are also shown (right graphs). Data are expressed as the mean value ± SEM (n = 3). (C) NEDD4L expression was down-regulated in HeLa cells as in (A) and autophagy was induced by starving cells for the indicated time periods. Levels of ULK1 and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (below graph). Data are expressed as the mean ± SEM (n = 3). Two different expositions (low and high) for ULK1 bands are shown. (D) HeLa cells were cotransfected with cDNAs coding for Myc-tagged ULK1WT or ULK1S1047A or ULK1K46I together with NEDD4LWT-HA in the presence or absence of MG132 or lactacystin for two different time periods (1 h and 2 h). Levels of ULK1, NEDD4L, and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (below graph). Data are expressed as the mean ± SEM (n = 3). (E) HeLa cells were cotransfected with a vector encoding 6xHIS-tag Ubiquitin and in some of them with NEDD4L-HA and autophagy was induced for 3 h in the presence of MG132. Protein extracts were prepared in a denaturing urea buffer and subjected to Ni-NTA purification. The amount of ubiquitylated NEDD4L copurified with 6xHIS-ubiquitin was evaluated by WB. (F) HeLa cells were treated with EBSS for the indicated time periods, and the levels of p-NEDD4L, NEDD4L, and ACTIN were detected by WB. (G) qPCR analysis of NEDD4L mRNA levels in HeLa cells incubated in EBSS medium for the indicated time periods. Data are expressed as mean ± SEM (n = 3). In B–D and G, data were analyzed by one-way ANOVA followed by Tukey post hoc test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
Figure 4.

NEDD4L degrades ULK1 during prolonged starvation. (A) NEDD4L expression was down-regulated in HeLa cells using specific RNAi oligonucleotides (oligos; siRNA NEDD4L#1) or unrelated oligos as negative control (siRNA CTRL). Densitometric analysis of ULK1 over ACTIN is also shown (right graph). Data are expressed as the mean value ± SD (n = 3), and statistical analysis was performed using an unpaired Student's t test. **, P < 0.01. (B) NEDD4L expression was down-regulated in HeLa cells using specific RNAi oligos targeting NEDD4L 3′UTR (siRNA NEDD4L#2) or unrelated oligos as negative control (siRNA CTRL). Some of them were then transfected with empty vector or NEDD4L-HA. Levels of ULK1, NEDD4L, and ACTIN were detected by WB. Densitometric analyses of both ULK1 and NEDD4L over ACTIN are also shown (right graphs). Data are expressed as the mean value ± SEM (n = 3). (C) NEDD4L expression was down-regulated in HeLa cells as in (A) and autophagy was induced by starving cells for the indicated time periods. Levels of ULK1 and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (below graph). Data are expressed as the mean ± SEM (n = 3). Two different expositions (low and high) for ULK1 bands are shown. (D) HeLa cells were cotransfected with cDNAs coding for Myc-tagged ULK1WT or ULK1S1047A or ULK1K46I together with NEDD4LWT-HA in the presence or absence of MG132 or lactacystin for two different time periods (1 h and 2 h). Levels of ULK1, NEDD4L, and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (below graph). Data are expressed as the mean ± SEM (n = 3). (E) HeLa cells were cotransfected with a vector encoding 6xHIS-tag Ubiquitin and in some of them with NEDD4L-HA and autophagy was induced for 3 h in the presence of MG132. Protein extracts were prepared in a denaturing urea buffer and subjected to Ni-NTA purification. The amount of ubiquitylated NEDD4L copurified with 6xHIS-ubiquitin was evaluated by WB. (F) HeLa cells were treated with EBSS for the indicated time periods, and the levels of p-NEDD4L, NEDD4L, and ACTIN were detected by WB. (G) qPCR analysis of NEDD4L mRNA levels in HeLa cells incubated in EBSS medium for the indicated time periods. Data are expressed as mean ± SEM (n = 3). In B–D and G, data were analyzed by one-way ANOVA followed by Tukey post hoc test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

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