Figure 3.

p32 interferes with the polyubiquitination of ULK1. (a) HeLa cells were transfected with HA-tagged WT or mutant ubiquitin constructs. Protein extracts were immunoprecipitated using anti-ULK1 antibody or rabbit IgG as a negative control (IgG). Immunoprecipitates were analyzed by western blotting using anti-ULK1 and anti-HA antibodies. (b) Hela cells expressing HA-Ubiquitin-k48 (HA-Ub-k48) were infected with the indicated p32-shRNAs. Cells were then treated with DMSO or MG132 (20 μM) for 6 h before being collected. Protein extracts were immunoprecipitated using anti-ULK1 antibody. The immunoprecipitates were subjected to western blotting with the indicated antibodies. (c) HeLa cells expressing HA-Ubiquitin-k63 (HA-Ub-K63) were infected with p32 shRNA lentiviral vector. Endogenous ULK1 protein was immunoprecipitated and immunoblotted with the indicated antibodies. Of note, ULK1 levels in the immunoprecipitation experiment do not change in this case in the p32 shRNA lane, owing to saturation of the anti-ULK1 antibody. (d) The indicated expression constructs were transiently transfected into HEK293T cells. Protein extracts were immunoprecipitated using anti-Myc antibody. The immunoprecipitates were subjected to western blotting with the indicated antibodies. (e) Hela cells stably expressing HA-AMBRA1 were infected with p32 shRNA lentiviral vector. Cell lysates were collected and analyzed by western blotting with the indicated antibodies. (f) Hela cells stably expressing HA-TRAF6 were infected with p32 shRNA lentiviral vector as in e. Cell lysates were collected and analyzed by western blotting with the indicated antibodies. (g) Hela cells infected with lentivirus bearing TRAF6-shRNA and p32-shRNA were collected. Protein extracts were subjected to western blotting with the indicated antibodies