Fig. 1

Polη interacts with OGT and is subject to O-GlcNAcylation predominantly at T457. a The nuclear extracts of 293T cells expressing Flag-Polη were immunoprecipitated. The indicated bands (black rectangles) were cut and analyzed via mass spectrometric analysis. b 293T cell lysates expressing Flag-Polη were immunoprecipitated and detected with anti-OGT and anti-Flag antibodies. The input included 2% of the cell lysate used. c U2OS cells were irradiated with UVC (15 J m⁻²) and harvested at different time points later. The chromatin fractions were extracted followed by immunoblotting with OGT and H3.1 antibodies. d U2OS cells were transfected with siOGT or siNC oligos. The OGT protein level was detected by western blot. The knockdown cells were transfected with GFP-Polη, irradiated with UVC (15 J m⁻²) and further incubated for 24 h. The proportions of GFP-Polη-expressing cells with >30 foci were determined. Data represent means ± SEM from three independent experiments. e 293T cells transfected with Flag-Polη or empty vector were incubated with Thiamet-G and glucose. The cell lysates were denatured and immunoprecipitated with anti-Flag M2 beads followed by immunoblotting with O-GlcNAc and Flag antibodies. NS: non-specific band. f 293T cells transfected with GFP-Polη were treated with Thiamet-G and different concentrations of glucose. The cell lysates were immunoprecipitated with GFP-Trap A followed by immunoblotting with O-GlcNAc and GFP antibodies. g 293T cells transfected with Flag-Polη or empty vector were treated with Thiamet-G and glucose. The cell lysates were immunoprecipitated with anti-Flag agarose followed by HCD-MS analysis. The tryptic peptide containing a HexNAc (+203.08 Da) was detected. The b- and y-type product ions were marked on the spectrum and also illustrated along the peptide sequence shown on top of the spectrum. h, i 293T cells transfected with the indicated GFP-Polη constructs were treated as in g. The cell lysates were immunoprecipitated with GFP-Trap A followed by immunoblotting with O-GlcNAc and GFP antibodies. SE short exposure, LE long exposure