Figure 3. NEDD4L ubiquitylates ULK1 and induces its degradation via proteasome. (A) HeLa cells were transfected with empty or NEDD4L-HA vectors; levels of ULK1, NEDD4L, and TUBULIN were detected by WB. Densitometric analysis of ULK1 over TUBULIN is also shown (right graph). Data are expressed as the mean ± SD (n = 3) and statistical analysis was performed using an unpaired Student's t test. **, P < 0.01. (B) HeLa cells were transfected with cDNAs coding for NEDD4LWT and NEDD4LCA HA-tagged proteins; ULK1, NEDD4L, and TUBULIN protein levels were detected by WB. Densitometric analysis of ULK1 over TUBULIN is also shown (right graph). Data are expressed as the mean ± SEM (n = 4). (C) HeLa cells were transfected with NEDD4L-HA in the presence or not of MG132 for 4 h. Levels of ULK1, NEDD4L, and TUBULIN were detected by WB. Densitometric analysis of ULK1 over TUBULIN is also shown (right graph). Data are expressed as the mean ± SEM (n = 3). (D) HeLa cells were cotransfected with a vector encoding a 6xHIS-tag ubiquitin and Myc-ULK1 in combination or not with NEDD4LWT and NEDD4LCA in the presence of MG132. Protein extracts were prepared in a denaturing urea buffer and subjected to Ni-NTA purification. The amount of ubiquitylated Myc-ULK1 copurified with 6xHIS-ubiquitin was evaluated by WB. (E) An in vitro ubiquitylation assay was performed by mixing immunopurified Myc-ULK1 and NEDD4L-HA, together with recombinant HIS-tag ubiquitin. ULK1 ubiquitylation is evaluated using an anti-Ubiquitin antibody to detect the incorporation of recombinant HIS-ubiquitin. The levels of ULK1 and NEDD4L were analyzed by WB. (F) HeLa cells were cotransfected independently with vectors encoding for 6xHIS-tag specific ubiquitin constructs and Myc-ULK1 in combination or not with HA-tagged NEDD4LWT or NEDD4LCA as a negative control. Protein extracts were prepared as in (D) and the amount of ubiquitylated ULK1 copurified with 6xHIS-Ubiquitin was evaluated by WB. The higher expression levels of NEDD4LCA construct are caused by the lack of activity on itself (Bruce et al., 2008). In B and C, data were analyzed by one-way ANOVA followed by Tukey post hoc test. *, P < 0.05.
Figure 3.

NEDD4L ubiquitylates ULK1 and induces its degradation via proteasome. (A) HeLa cells were transfected with empty or NEDD4L-HA vectors; levels of ULK1, NEDD4L, and TUBULIN were detected by WB. Densitometric analysis of ULK1 over TUBULIN is also shown (right graph). Data are expressed as the mean ± SD (n = 3) and statistical analysis was performed using an unpaired Student's t test. **, P < 0.01. (B) HeLa cells were transfected with cDNAs coding for NEDD4LWT and NEDD4LCA HA-tagged proteins; ULK1, NEDD4L, and TUBULIN protein levels were detected by WB. Densitometric analysis of ULK1 over TUBULIN is also shown (right graph). Data are expressed as the mean ± SEM (n = 4). (C) HeLa cells were transfected with NEDD4L-HA in the presence or not of MG132 for 4 h. Levels of ULK1, NEDD4L, and TUBULIN were detected by WB. Densitometric analysis of ULK1 over TUBULIN is also shown (right graph). Data are expressed as the mean ± SEM (n = 3). (D) HeLa cells were cotransfected with a vector encoding a 6xHIS-tag ubiquitin and Myc-ULK1 in combination or not with NEDD4LWT and NEDD4LCA in the presence of MG132. Protein extracts were prepared in a denaturing urea buffer and subjected to Ni-NTA purification. The amount of ubiquitylated Myc-ULK1 copurified with 6xHIS-ubiquitin was evaluated by WB. (E) An in vitro ubiquitylation assay was performed by mixing immunopurified Myc-ULK1 and NEDD4L-HA, together with recombinant HIS-tag ubiquitin. ULK1 ubiquitylation is evaluated using an anti-Ubiquitin antibody to detect the incorporation of recombinant HIS-ubiquitin. The levels of ULK1 and NEDD4L were analyzed by WB. (F) HeLa cells were cotransfected independently with vectors encoding for 6xHIS-tag specific ubiquitin constructs and Myc-ULK1 in combination or not with HA-tagged NEDD4LWT or NEDD4LCA as a negative control. Protein extracts were prepared as in (D) and the amount of ubiquitylated ULK1 copurified with 6xHIS-Ubiquitin was evaluated by WB. The higher expression levels of NEDD4LCA construct are caused by the lack of activity on itself (Bruce et al., 2008). In B and C, data were analyzed by one-way ANOVA followed by Tukey post hoc test. *, P < 0.05.

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