Figure 4.

Overexpression of UBE3C inhibits autophagy under starvation conditions. (A-B) analysis of the effect of UBE3C on starvation induced autophagy. 293T cells transfected with his-UBE3C for 48 h or stably expressing sh-UBE3C were incubated with EBSS for 1 h with or without 0.5 μM of bafilomycin A₁ (baf) for 3 h, and then analyzed by immunoblotting with indicated antibodies. Quantification of LC3-II:GAPDH ratios were performed. n = 3, *P < 0.05,**P < 0.01. (C) the amount of endogenous LC3 puncta was affected by UBE3C. HeLa cells were transfected with his-UBE3C or sh-UBE3C plasmids as indicated for 48 h, then incubated with EBSS for 1 h. More than 50 cells were counted for the quantification of LC3 puncta per experimental condition. n = 4, **P < 0.01, ***P < 0.001. Scale bar: 10 μm. (D) analysis of the effect of UBE3C on nonradioactive quantification of autophagic protein degradation with l-azidohomoalanine labeling. HeLa cells were transfected with or without sh-UBE3C or his-UBE3C plasmids for 4 h and labeled with AHA for 20 h, then cells transfected with his-UBE3C were starved in EBSS for another 12 h. Fluorescence tagged of AHA-labeled proteins with the click reaction and then relative fluorescence intensity of the cells were analyzed by flow cytometry. In this chart, the lower intensity means the higher protein degradation activity. n = 5, ***P < 0.001.