RNF111 promotes UV-induced ubiquitylation of XPC. (A) U2OS or U2OS/Strep-HA-ubiquitin cells transfected with control (−) or RNF111 siRNAs were exposed or not exposed to UV and collected 1 h later, and XPC ubiquitylation was analyzed by immunoblotting Strep-Tactin pull-downs of whole-cell extracts (WCE) with the XPC antibody. (B) HeLa/FLAG-SUMO2 cells transfected with control (−) or RNF111 siRNAs and left untreated or induced to express FLAG-SUMO2 by addition of doxycycline (DOX) were exposed or not exposed to UV and collected 1 h later. Cells were lysed under denaturing conditions, and XPC SUMOylation was analyzed by immunoblotting of FLAG IPs with XPC antibody. (C) U2OS/Strep-HA-ubiquitin cells transfected with empty vector (−) or FLAG-RNF111 plasmid were exposed or not exposed to UV and collected 1 h later. XPC ubiquitylation was analyzed as in A. (D) XPC ubiquitylation in U2OS/Strep-HA-ubiquitin cells depleted of RNF111 or Ubc13 was analyzed as in A. Ubc13 knockdown efficiency is shown in Fig. S3 D. (E) Extracts of U2OS/GFP-RNF111 cells collected at the indicated times after UV radiation were subjected to GFP IP followed by immunoblotting with XPC antibody. (F) Extracts of U2OS cells incubated with or without MG132, exposed to UV 30 min later, and collected at the indicated times after UV were analyzed by immunoblotting with the RNF111 antibody. Asterisks denote a nonspecific band. MM, molecular mass.