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. 2017 Apr 5;8(22):36423–36437. doi: 10.18632/oncotarget.16855

Figure 4. Parkin facilitates ssDNA generation and efficient RPA recruitment in response to UV radiation.

Figure 4

(A–B) WT and Parkin−/− cells were irradiated with 15 J/m2 UV and further incubated for 2 h. Cells were pre-extracted with 0.5% Triton for 10 min and then fixed, immunostained with anti-RPA32 antibody and Hoechst-stained. (A) Representative images of cells stained with Hoechst or antibody against RPA32 after UV radiation. (B) Quantification of the percentage of cells with more than 20 RPA32 foci. Error bars represent SD. t-test, n = 3. (C) WT and Parkin−/− cells were irradiated with 15 J/m2 UV and further incubated for 2 h. The chromatin fraction were harvested and separated by SDS-PAGE. The levels of RPA32 were detected by western blot with an anti-RPA32 antibody. The levels of RPA32 and PCNA in whole-cell lysates (WCL) were also detected. (D) Parkin−/− cells complemented with Flag-Parkin or empty vector were treated and examined as in (C). (E–F) WT and Parkin−/− cells were incubated with 10 μM BrdU for 48 h before irradiated with 15 J/m2 UV, and further incubated for 2 h. Cells were pre-extracted with 0.5% Triton and immunostained with anti-BrdU antibody and Hoechst-stained. (E) Representative images of cells stained with Hoechst or antibody against BrdU after UV radiation. (F) Quantification of the percentage of cells with more than 20 BrdU foci. Error bars represent SD. t-test, n = 3.