FIGURE 1.

DNA damage induces an increase in nuclear levels of Rad51 in Brca2-proficient and Brca2-deficient cells. HeLa (A), HCT116 (B), and Capan-1 (C) cells grown at 37 °C were harvested at the indicated times following exposure to 2 Gy of IR and fractionated as described under “Experimental Procedures” to yield cytoplasmic (Cyto), nucleoplasmic (Nuc), and chromatin (Chrom) samples. D and E, HCT116 and Capan-1 cells, respectively, were treated with cycloheximide (CHX; 20 μm) 1 h prior to exposure to 2 Gy of IR. A portion of each fraction (30 μg of total protein) was loaded onto 4–12% SDS-polyacrylamide gels, and Western blots were developed using a mouse anti-Rad51 monoclonal antibody. Blots were also developed using the following markers as loading controls: glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cytoplasmic), Sam68 (nucleoplasmic), and fibrillarin (chromatin). F, changes in levels of nuclear Rad51 as a function of time after IR treatment in A–E were quantified as described under “Experimental Procedures.” The data shown are representative of the results of at least three separate experiments, and the S.D. observed with quantification was <20%.