Fig. 4: OGA mediates deglycosylation of NONO and the Ku70/80 complex.

A The O-GlcNAcylation of NONO and the Ku70/80 complex were induced in response to DNA damage. 293T cells were treated with 10 Gy of IR and after 1 h cells were lyzed with NETN300 buffer. Cell extracts were examined by IP and western blot with indicated antibodies. The histogram shows the relative level of the O-GlcNAcylated NONO, Ku70, Ku80 signal. B Time course analyses of O-GlcNAcylation of NONO and the Ku70/80 complex events with mock or the TMG treatment following IR treatment using Western blotting. 293T cells were treated with mock or TMG and then exposed to 10 Gy of IR. Cell extracts were examined by IP and western blot with indicated antibodies. C The HAT domain of OGA recognizes O-GlcNAcylated NONO and the Ku70/80 complex following DNA damage. 293T cells were transfected with SFB-C-OGA and then two-step Co-IP assay was performed. Streptavidin beads were used to bind the HAT domain of OGA (C-OGA) partners in the first Co-IP assay. Anti-NONO, anti-Ku70/80 antibodies were used to IP target proteins in the second Co-IP assay. Western blot assay was performed with indicated antibodies. D Time course analyses of the deglycosylation of NONO and the Ku70/80 complex by full-length OGA or N-OGA following IR treatment using western blot. Cells were treated with siOGA and rescued with FL OGA or truncated OGA. Then these cells were exposed to 10 Gy of IR. Cell extracts were examined by IP and western blot with indicated antibodies.