RNF111 has STUbL activity in the presence of Ubc13–Mms2. (A) MS-based analysis of RNF111-interacting proteins. U2OS and U2OS/GFP-RNF111 cells were grown in light and heavy SILAC medium, respectively. GFP-RNF111 and associated proteins enriched on GFP-Trap resin were analyzed by MS. Plot shows z scores (from SILAC heavy/light ratios) and total intensity of identified proteins. RNF111, Ubc13 (UBE2N), and Mms2 (MMS2) are highlighted. See also Fig. S1 (A and B). (B) U2OS cells were cotransfected with indicated combinations of GFP-RNF111 and Strep-HA-Ubc13 plasmids. Whole-cell extracts (WCE) were subjected to Strep-Tactin pull-down followed by immunoblotting with GFP and HA antibodies. (C) U2OS cells transfected with nontargeting (control [CTRL]) or RNF111 siRNAs were collected 72 h later and processed for immunostaining (top) or immunoblot (bottom) with RNF111 antibody. Asterisk indicates a nonspecific band. Bar, 10 µm. (D) Extracts of U2OS cells sequentially transfected with RNF111 siRNA and S-FLAG-Strep–tagged RNF111 (SFS-RNF111) plasmids were subjected to Strep-Tactin pull-down. Bound complexes were incubated with ubiquitylation reaction mixture containing E1, Ubc13–Mms2 complex, and HA-ubiquitin as indicated and washed extensively, and RNF111 E3 ligase activity was analyzed by immunoblotting with the HA antibody. (E) As in D, except that ubiquitylation reactions were performed in the presence or absence of poly-SUMO2 (3–8) chains followed by immunoblotting with HA and SUMO2 antibodies. MM, molecular mass.