Figure 2.

UBE3C assembles K33-branched ubiquitin chain on ATG4B. (A) analysis of the effect of UBE3C on the ubiquitination level of ATG4B. The ubiquitinated proteins were pulled down by anti-flag-gel from 293T cells transfected with flag-vector, or flag-ATG4B, HA-Ub, UBE3C, and sh-UBE3C and analyzed by western blotting with indicated antibodies. The transfection lasted 24 h, following cells were incubated with MG132 (10 µM) for 6 h. Ratios of ubiquitinated ATG4B:Input flag-ATG4B were quantified. n = 3, ***P < 0.001, ****P < 0.0001. (B) analysis of the effect of UBE3C on the ubiquitination level of ATG4B[K0]. 293T cells were transfected with flag-vector, flag-ATG4B[K0], HA-Ub, UBE3C, and sh-UBE3C as indicated, after 24 h, cells were incubated with MG132 (10 µM) for 6 h, cell lysates were subjected to IP with anti-flag-gel and then analyzed by western blotting. Ratios of ubiquitinated ATG4B:Input flag-ATG4B were quantified. n = 3, ns: no significance. (C) analysis of the types of ATG4B ubiquitin chain that affected by UBE3C. The ubiquitinated proteins were pulled down by anti-flag-gel under denaturing conditions from 293T cells transfected with indicated constructs and analyzed by western blotting with indicated antibodies. After 24 h transfection, cells were incubated with MG132 (10 µM) for another 6 h. The amounts of different ubiquitin constructs used for transfection were adjusted to allow an equal level of ATG4B ubiquitination among the groups without UBE3C transfection. (D) 293T cells were transfected with UBE3C plasmids for 24 h, following cells were incubated with MG132 (10 µM) for 6 h, cell lysates were subjected to IP and then analyzed by western blotting. The ubiquitinated endogenous ATG4B was detected using the anti-ub-K33 antibody. Ratios of ATG4B with K33 ub chain:endogenous ATG4B were quantified. n = 3, *P < 0.05.