Figure 1.

Identification of E3 ligase UBE3C as a novel protein binds to ATG4B. (A) ubiquitination of ATG4B was detected by immunoprecipitation (IP). 293T cells were transfected with flag-ATG4B or flag-vector respectively together with HA-Ub. After 24 h, cells were incubated with 10 µM of MG132 for 6 h, cell lysates were subjected to IP with anti-flag-gel and then analyzed by SDS-PAGE, and ubiquitinated ATG4B were detected using an anti-HA antibody. (B) a total of 79 probable ATG4B-interacting proteins under the ubiquitination conditions. The heat map showed proteins appeared at least three times in the experimental group (flag-ATG4B and HA-Ub plasmids co-overexpressed in 293T cells) from six repeats of tandem mass spectrometry. (C) HeLa cells transfected with GFP-ATG4B and mCherry-UBE3C plasmids were incubated on a confocal dish, then treated with 0.1% DMSO or MG132 (10 μM) for 6 h. Cells were examined by immunofluorescence microscopy to detect the colocalization of mCherry-UBE3C and GFP-ATG4B. Scale bar: 10 μm. (D) interaction of endogenous UBE3C and endogenous ATG4B were detected without MG132 treatment by western blotting. (E) GST-ATG4B or GST control protein (5 µg) were incubated with GST agarose gel in TBST buffer at room temperature. After 1 h of incubation, HECT segment of UBE3C (5 µg) was added, and thoroughly mixed in vibrating mixer for overnight incubation at 4°C. Then GST-gel was collected and loaded for western blotting. ATG4B and UBE3C levels were detected by GST and UBE3C antibodies. All representative blots are from at least 3 independent experiments. (F) cell lysates of 293T cells transfected with GFP-ATG4B truncations were incubated with GFP-agarose gel overnight at 4°C. Immunoprecipated ATG4B truncations and endogenous UBE3C were detected by western blotting with GFP and UBE3C antibodies.