Fig. 4: XRCC4 is O-GlcNAcylated on Thr308.

A HCT116 cells were transfected with FLAG-XRCC4 or Myc-OGT for 24 h. The cell lysates were immunoprecipitated using FLAG beads. Exogenous O-GlcNAcylation of XRCC4 was detected by immunoblotting with RL2 antibody. For co-immunoprecipitation, the immunoprecipitated lysates were immunoblotted with Myc antibody. B, C HCT116 cells were transfected with B Myc-OGT or C V5-OGA for 24 h. The cell lysates were immunoprecipitated using the XRCC4 antibody. Endogenous O-GlcNAcylation of XRCC4 was detected by immunoblotting with RL2 antibody. B For co-immunoprecipitation, the immunoprecipitated lysates were immunoblotted with Myc antibody. D Putative O-GlcNAc sites of XRCC4 were identified by EThcD-MS/MS. O-GlcNAcylated XRCC4 peptides, 297-ENSRPDSSLPEtSK-310, is shown. E A schematic drawing of the putative XRCC4 O-GlcNAcylation sites, including Ser303, Ser304, Thr308, and Ser309, based on mass spectrometry data. F HCT116 cells were transiently transfected with each indicated FLAG-XRCC4 point mutant. The cell lysates were immunoprecipitated using FLAG beads. O-GlcNAcylation of each FLAG-XRCC4 point mutant was detected by immunoblotting with RL2 antibody. O-GlcNAcylated FLAG-XRCC4 was normalized to immunoprecipitated FLAG-XRCC4 (n = 4). Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t-test (***P < 0.001). G Cross-species sequence alignment of XRCC4.