ULK1 interacts with the E3 ligase NEDD4L. (A) Protein extracts from HeLa cells were immunoprecipitated using anti-NEDD4L, anti-ULK1 antibodies and with IgG as a negative control. NEDD4L and ULK1 were analyzed by WB. The two NEDD4L bands correspond to two different spliced isoforms. (B) HeLa cells were transiently transfected with Myc-ULK1 and then grown in normal or EBSS medium for 1 and 3 h, respectively. Protein extracts were immunoprecipitated using an anti-Myc antibody or with IgG as a negative control; Myc-ULK1 and NEDD4L were analyzed by WB. (C) HeLa cells were grown either in normal or in EBSS medium for 90 min, respectively. Protein extracts were immunoprecipitated using an anti-NEDD4L antibody or with IgG as a negative control. ULK1 and NEDD4L analyzed by WB. The band density ratio of immunoprecipitated ULK1, relative to immunoprecipitated NEDD4L normalized on the input amount of ULK1, is analyzed (n = 3). Data are expressed as the mean ± SD, and statistical analysis was performed using an unpaired Student's t test. **, P < 0.01. (D) Scheme of NEDD4L full length and fragments showing the N-terminal C2 domain, four WW domains (black boxes), and the HECT domain. HeLa cells were cotransfected with vectors encoding Myc-ULK1 together with NEDD4L-HA fragments encoding the WW1, WW2, and WW3-4 domains, respectively. Protein extracts were immunoprecipitated using an anti-HA antibody; Myc-ULK1 and NEDD4L-HA fragments are analyzed by WB. Asterisk represents unspecific bands.