Fig 3.
CAPNS1 perturbs PCNA ubiquitination. CAPNS1 or LACZ was depleted from U2OS cells using specific siRNAs, and 72 h later, the cells were either left untreated or irradiated with 30 J/m2 UV. The cells were fixed 1, 5, and 20 h later, and Triton-insoluble PCNA was detected by immunofluorescence. (a) Typical images of PCNA foci. (b) Average number of PCNA foci at different time points after irradiation. Two hundred nuclei were analyzed for each sample. The error bars represent standard deviations from three independent experiments. (c) HA-CAPNS1-expressing U2OS cells, U2OS cells infected with pRetro Super carrying sh-CAPNS1 or an empty pRetro Super vector were irradiated or not with 30 J/m2 UV light, and the respective lysates were prepared 1, 3, and 5 h postdamage to monitor the PCNA ubiquitination (Ub-PCNA) level. (d) U2OS cells infected with pRetro Super carrying sh-CAPNS1 or an empty pRetro Super vector were irradiated or not with 15, 30, and 60 J/m2 UV light, and the respective lysates were prepared 1, 5, and 20 h after irradiation to monitor PCNA ubiquitination by Western blotting. Longer and shorter exposures are shown. (e) U2OS cells, stably expressing hyperstable GFP-USP1 mutated in its autocleavage site GFP-USP1(GG670/671AA) or catalytically inactive GFP-USP1C90S (f) were transfected with LACZ- or CAPNS1-specific siRNA and 72 h later irradiated with 30 J/m2 UV or left untreated. The lysates were collected at various time points (1, 3, 5, and 20 h postdamage) and utilized to monitor endogenous PCNA ubiquitination by Western blotting. Longer and shorter exposures are shown.