Fig. 2: RNF168 ubiquitinates H2A variants at a specific lysine residue.

a Schematic diagram of the lysine residue distribution of H2AZ. b H2AZ C-terminus is the major Ub-acceptor. SFB-H2AZ wildtype and mutants were transfected in HEK293T cells for 24 h, harvested with SDS-PAGE sample buffer, and followed by western blot analysis using Flag antibody and tubulin as loading control. Repeated two times independently with similar results. c RNF168 specifically ubiquitinates H2AZ at K15. Co-transfection of Myc-RNF168 and SFB-H2AZ wildtype and mutants with lysine to arginine mutation in HEK293T cells as indicated. Repeated three times independently with similar results. d–f RNF168 ubiquitinates H2A variants at a specific lysine residue. SFB-H2AZ, SFB-macroH2A1, and SFB-macroH2A2 wildtype and mutants were co-transfected with Myc-RNF168 in HEK293T cells followed by western blot analysis using antibodies as indicated. Repeated at least three times independently with similar results. g–i RNF168 is required for site-specific ubiquitination of H2A variants. HEK293T cells with stable expression of CMV-SFB-H2AZ 9K to R-R15K, CMV-SFB-MacroH2A1 8K to R-R11K, EF1α -SFB-MacroH2A2 8K to R-R11K were transfected with siRNAs targeting RNF168. Cells were harvested and pulled down using streptavidin agarose. Pull-down samples were analyzed by western blot with indicated antibodies. Repeated four times for g-h and two times for i with similar results. Source data are provided as Source Data file.