Fig. S5.

Ubiquitylation of TRC35 in cells expressing Bag6 mutants. (A) IP of TRC35 in Bag6^−/− 293T cells cotransfected with plasmids encoding TRC35⋅FLAG (WT), Bag6⋅GFP (WT or mutants), and HA⋅ubiquitin. Anti-GFP Ab was used for the first IP. Anti-FLAG Ab was used for the second IP in denaturing conditions. TRC35 ubiquitylation was assessed by immunoblotting with anti-HA Ab. The exposure times were adjusted to improve visibility of the reactive bands. (B) IP was carried out as in A in Bag6^−/− 293T cells cotransfected with plasmids encoding TRC35⋅FLAG (WT) and Bag6⋅GFP (WT or mutants). TRC35 ubiquitylation was assessed by immunoblotting with anti-FLAG Ab. The exposure times were adjusted to improve visibility of the reactive bands. (C) The cell extract used for IP was immunoblotted for Bag6, TRC35, and HA⋅ubiquitin with Bag6 Ab, FLAG Ab, and HA Ab, respectively.