Fig. 3.

Ubiquitylation of TRC35 in cells expressing Bag6 mutants is dependent on RNF126. (A) Bag6^−/− 293T cells cotransfected with plasmids encoding TRC35•FLAG (WT), Bag6⋅GFP (WT or mutant), and HA•Ub and treated with 10 µM MG132. The cell extracts were subject to two rounds of IP with anti-GFP Ab then with anti-FLAG Ab in denaturing conditions. Ubiquitylation was assessed by immunoblotting with HA Ab. (B) Bag6^−/− 293T cells cotransfected with plasmids encoding TRC35•FLAG (WT), Bag6•GFP (WT or mutants), and HA⋅Ub. The cells were simultaneously treated with 10 µM MG132 and siRNAs against RNF126. The cell extracts were subject to two rounds of denaturing IP with anti-GFP Ab and then with anti-FLAG Ab. TRC35 ubiquitylation was assessed by immunoblotting with HA Ab.