hRAD51C forms nuclear foci in response to IR. (A) Western blot detection of hRAD51C. An immunoblot of extracts prepared from irradiated and nonirradiated HeLa and U2OS cells was probed with anti-hRAD51C antibody. Recombinant hRAD51C-His₁₀, migrating more slowly because of its epitope tag, served as a control. (B) Immunofluorescence detection of RAD51C in paraformaldehyde-fixed HeLa and U2OS cells. Irradiated (10 Gy) and nonirradiated cultures were stained with anti-hRAD51C monoclonal antibody (red) 2 h after treatment. DNA was counterstained with DAPI (blue). (C) siRNA-mediated reduction of RAD51C expression. Cells were transfected with control or RAD51C siRNA and irradiated 48 h after transfection. Cell extracts, prepared 2 h after irradiation, were analyzed by Western blotting. (D) siRNA-mediated reduction of RAD51C foci. As in C, but cells were fixed 2 h after IR and stained with anti-hRAD51C monoclonal antibody (red). Image acquisition parameters were identical for the three samples. (E) Partial colocalization of RAD51C and RAD51 in irradiated HeLa cells stained with anti-RAD51C (red) and anti-RAD51 antibodies (green). Nuclei displaying only RAD51C or RAD51 foci or containing both types of foci are labeled with a red, green, or yellow circle, respectively. (F) Enlarged image of a cell containing both RAD51C (red) and RAD51 foci (green). Bars, 10 µm.