RNF111 ubiquitylates XPC in a SUMOylation-dependent manner. (A) Outline of in vitro SUMO-binding and STUbL assays. XPC-GFP expressed in U2OS cells was immunopurified on GFP-Trap resin and subjected to in vitro SUMOylation. After washing, the XPC-GFP–containing beads were incubated with extracts of cells transfected or not transfected with S-FLAG-Strep-RNF111 (SFS-RNF111) constructs, washed again, and processed for immunoblotting (IB) of bound SFS-RNF111 with FLAG antibody (i) or subjected to in vitro ubiquitylation followed by washing and immunoblotting with the HA antibody to analyze ubiquitin ligase activity (ii). (B) SUMOylation-dependent binding of RNF111 to XPC, analyzed as described in A. (C) Analysis of SUMOylation-dependent XPC ubiquitylation by RNF111 was performed as described in A. MM, molecular mass.