Fig. 4.

RPA2 hyperphosphorylation is required for the HR stimulated by HU but not for HR induced by I-Sce-I. (A) RPA2 hyperphosphorylation is specifically critical for HR after replication arrest. The frequencies of HR were calculated in cells expressing RPA2-WT or RPA2-A after HU treatment by flow cytometry. Results are means from three independent experiments with standard deviation. (B) Cells expressing phosphorylation-defective RPA2 show similar HR frequency induced by I-Sce-I expression in comparison to cells with wild-type RPA2. The HR frequency was measured by flow cytometric analyses (see Materials and Methods) in cells expressing RPA2-WT or RPA2-A. Results are means from three independent experiments, with standard deviation. (C) Immunoblot analysis of RPA2 hyperphosphorylation expression in MCF7 cells with I-Sce-I plasmid transfection or HU and Thy. Cell lysates were harvested 24 h after I-Sce-I transfection or after HU and Thy treatment. The doses used for HU and Thy are same as Figure 1A. Proteins were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and visualized by immunoblot with anti-RPA2-p4/8 antibody or anti-RPA2 antibody (Abcam). (D) Foci of phosphorylated RPA2 in cells with I-Sce-I transfection or HU treatment. The cells were fixed 24 h after I-Sce-I transfection or 4 h after HU treatment. Fixed cells were stained by a specific anti-RPA2 phosphorylation antibody and anti-RPA2-p4/8 antibody. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).