Fig. 5

UBE2T binds with H2AX/γH2AX, and promotes CHK1 activation, DDR and HCC radioresistance via H2AX/γH2AX. a FLAG-UBE2T complexes were purified from 293 T cells transfected with the indicated plasmids and then treated with or without IR (4 Gy). The indicated bands were the distinct bands stained by silver stain and then identified by mass spectrometry. b Immunoprecipitation of H2AX with UBE2T from 293 T cells transfected with FLAG-UBE2T after IR (4 Gy). c Immunoprecipitation of UBE2T with H2AX from 293 T cells transfected with plasmids encoding FLAG-H2AX was analyzed by immunoblotting after IR (4 Gy). d-e The MHCC-97H cells treated with or without IR and made for immunoprecipitation by using anti-UBE2T antibody (d) or anti-H2AX antibody (e). f MHCC-97H cells were transfected with FLAG-UBE2T and were treated with IR, FLAG-UBE2T (green), or γH2AX (red) were analyzed with immunofluorescence assay. Scale bar: 20 μM. g UBE2T overexpressing or control MHCC-97H cells transfected with H2AX siRNA or control siRNA, and then treated with IR (4 Gy). Total cell lysates were analyzed by immunoblotting analysis. h Cells were treated as panel g. The cytosol and chromatin fractionation of the cells were isolated and analyzed by immunoblotting. i Cells were treated as panel g. Cells were harvested at indicated timepoints, and stained for CHK1, representative images of immunofluorescence staining were shown. Scale bar: 20 μM. j Cells were treated as panel g. Cell cycle distribution was analyzed by flow cytometry. The average numbers of % cells in the G2 phase are shown above the bars. k Cells were treated as panel g. Quantification of comet assays is shown. l Cells were treated as panel g. Cell survival was assessed by CCK-8 assay. Data represent the mean ± SD. In (j) and (k), ns, not significant, *P < 0.05 and **P < 0.01, by one-way ANOVA. In (l), **P < 0.01, by two-way ANOVA