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. 2019 Oct 15;8(10):1258. doi: 10.3390/cells8101258

Figure 4.

Figure 4

WIP1 delays recruitment of BRCA1 and dephosphorylation of 53BP1 at T543. (A) Co-immunoprecipitation of WIP1 and 53BP1. HEK293 cells were transfected with either empty GFP or GFP-WIP1, subjected to immunoprecipitation using GFP-Trap 24 h after transfection and by Western blotting with 53BP1 antibody. Ponceau staining with indicated positions of GFP (empty arrowhead) and GFP-WIP1 (full arrowhead) are shown. (B) HEK293 cells transfected with EGFP or EGFP-WIP1 were exposed to 3 Gy of IR, collected at indicated times and proteins were immunoprecipitated by GFP Trap. (C) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points after IR and stained with p53BP1 T543 antibody. Click chemistry was used to visualize EdU. Mean of median total intensity +/− SD is plotted. (D) Western blot analysis of whole cell lysates of U2OS cells transfected with GAPDH or PP4C siRNA in response to irradiation and/or WIP1 inhibitor. (E) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were transfected with control or PP4C siRNA 2 days before irradiation. Cells were processed and analyzed as in C. (F) Quantification of RPA2 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i were pulse-labeled with EdU for 30 minutes before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with RPA2 and RAD51 antibodies. Click chemistry was used to visualize EdU. Mean of median foci number +/− SD is plotted. (G) Quantification of RAD51 foci in replicating (EdU+) cells after irradiation as in F.