Fig. 3

T457A impairs Polη removal from chromatin after UV irradiation. a U2OS cells transfected with WT or mutated GFP-Polη constructs were 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. b, c Flag-UFD1 b or Flag-NPL4 c and GFP-Polη (WT or T457A) were transfected into 293T cells. The lysates were immunoprecipitated using anti-Flag M2 beads. The inputs and immunoprecipitates were examined via western blotting using anti-GFP and anti-Flag antibodies. d 293T cells were transfected with WT or mutant Flag-Polη as well as HA-Ub or HA empty vector. The cell lysates were immunoprecipitated with anti-Flag M2 beads under denaturing condition, followed by immunoblotting with anti-HA, anti-K48-Ubiquitin, and anti-Flag antibodies. e Mutation frequency in damaged (400 J m⁻² UVC) supF plasmid was determined. Data represent means ± SEM from three independent experiments. ns not significant. f XP30RO cells stably expressing GFP, WT or T457A GFP-Polη and MRC5 cells were irradiated with the indicated doses of UVC and further incubated in medium supplemented with 0.4 mM caffeine for 7–10 days. The number of cell clones was determined. Surviving fraction was expressed as a percentage of mock-treated cells. Experiment was repeated three times, giving similar results. The representative curve is shown. Error bar: s.d., n = 3