Fig. 5

RNF152 targets TSPAN12 for ubiquitination and degradation. a The cell lysates of MG132-treated HuH6 cells were subjected to immunoblot with indicated antibodies. b The cell lysates of HepG2 cells transfected with EV or Flag-RNF152 were subjected to immunoblot with indicated antibodies. c HepG2 cells transfected with control or Flag-RNF152 plasmids were treated with 20 μM MG132 for 4 h. The cell lysates were immunoprecipitated by Tandem Ubiquitin Binding Entity 2 (TUBE2) resin for ubiquitinated proteins enrichment and immunoblotted as indicated. d 293 T cells were transfected with the indicated plasmids for 36 h. After immunopurification with anti-HA resin, in vitro ubiquitylation of TSPAN12 was performed in the presence of E1, E2s, Flag-RNF152 and ubiquitin (Ub). Samples were incubated at 37℃ and subjected to immunoblot with indicated antibodies. e The cell lysates of HuH6 cells transfected with con-shRNA or RNF152-shRNA were subjected to immunoblot with indicated antibodies. f HuH6 cells transfected with con-shRNA or RNF152-shRNA were treated with 40 μM cycloheximide (CHX) for 0, 2, 4, 8, 10 h. The cell lysates were subjected to immunoblot as in g. Statistic results of western blotting analysis were obtained by image J software and normalized to actin intensities. Error bars indicate the means ± SD., n = 3. g HuH6 cells transfected with con-shRNA or RNF152-shRNA were treated with 20 μM MG132 for 4 h. The cell lysates were immunoprecipitated by TUBE2 resin for ubiquitinated proteins enrichment and immunoblotted as indicated