RAD51C is required for IR-induced CHK2 phosphorylation. (A) U2OS cells were transfected with control or RAD51C siRNA and irradiated (10 Gy) 48 h after transfection. After 2 h, extracts were prepared and immunoblotted as indicated. (B) As in A, but cells were treated with 1 Gy of IR, and protein samples were analyzed after 0.5, 1, and 2 h. (C) As in B, but using HeLa cells. (D) U2OS cells were transfected with a control siRNA or two different siRNAs against XRCC3 and irradiated (1 Gy) 48 h after transfection. After 2 h, extracts were prepared and immunoblotted as indicated. (E) HCT116 Chk2^−/− cells were treated with 10 Gy of irradiation and fixed 2 h after. Cells were stained with anti-RAD51C (red) and anti-RPA antibodies (green). (F) Model for RAD51C action in response to DSBs in S/G2. RAD51C is recruited by RPA to break sites, where it facilitates CHK2-mediated cell cycle arrest and assembly of the HR machinery. Bar, 10 µm.