Figure 3.
UBE3C mediates ATG4B ubiquitination at Lys119. (A) analysis of the effect of UBE3C on the ubiquitination level of different K-to-R mutants. 293T cells were transfected with different K-to-R mutants of flag-ATG4B together with HA-Ub and UBE3C with MG132 (10 µM) for another 6 h. Cell lysates were subjected to IP and then analyzed by western blotting. (B) in vitro ubiquitination assay was performed with 5 μg of purified his-ATG4B, His-ATG4BK119R, HECT and other required reagents as shown in the methods section. The mixture was incubated at 37°C for 120 min. Ubiquitination level was detected by anti-ub antibody and quantified. n = 3, ***P < 0.001. (C) analysis of the effect of UBE3C on the protein level of ATG4B. 293T cells or MEF cells were transfected with plasmids as indicated. Cell lysates were analyzed by western blotting with indicated antibodies.