Journal:

Article Title: Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells

doi:

Figure Lengend Snippet: Constitutive and X-ray-induced levels of the Rad51 paralogs in different human cell lines. Western blot analysis using different antibodies was used to determine the constitutive level of the Rad51 paralogs and of hRad51, and the level of these proteins 4 and 8 h after treatment with 10 Gy X-rays. P53 was used as a control for the X-ray treatment (see text), and either β-tubulin or transcription factor QM was used as loading standards for each western blot (only one representative blot for each is shown). Unlike Figure 1, the detection of XRCC3 here was done using an antibody from Novus that only weakly recognizes the recombinant XRCC3-(His)6 protein (see text).

Article Snippet: For the detection of hRad51 we used polyclonal rabbit anti-hRad51 antibody (1:3000, PC-130; Calbiochem) or monoclonal mouse anti-hRad51 antibody (1:1000, NB100-148; Novus Biologicals).

Techniques: Western Blot, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: 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 cyclo- heximide (CHX; 20 M) 1 h prior to exposure to 2 Gy of IR. A portion of each fraction (30 g of total protein) was loaded onto 4–12% SDS-polyacrylamide gels, andWesternblotsweredevelopedusingamouseanti-Rad51monoclonalantibody.Blotswerealsodevelopedusingthefollowingmarkersasloadingcontrols: 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%.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: FIGURE 2. Levels of Capan-1 nuclear Rad51 increase in an IR dose-de- pendent manner. Capan-1 cells exposed to 5 or 8 Gy of IR were grown at 37 °C for 2 h. Cells were harvested and fractionated as described under “Experimental Procedures,” and portions of the cytoplasmic (Cyto) and nucle- oplasmic (Nuc) fractions (25 g of total protein) were loaded onto 4–12% SDS-polyacrylamide gels. Western blots were developed using a mouse anti- Rad51 monoclonal antibody, and levels of cytoplasmic and nuclear Rad51 were quantified as described under “Experimental Procedures” (supplemen- tal Table 1). The blot shown is representative of four separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: FIGURE 4. Rad51C depletion decreases the steady-state level of nuclear Rad51 and diminishes its DNA damage-inducednucleartransportinBrca2-proficientandBrca2-deficientcells.HeLa(A)andCapan-1(B) cells were transfected with a nonspecific () or Rad51C-specific () siRNA pool (SMARTpool), grown for 42 h at 37 °C, exposed to 6 Gy of IR, and harvested 2 h later. Cytoplasmic (Cyto) and nuclear (Nuc) fractions were analyzed by Western blotting using an anti-Rad51 monoclonal antibody. Changes in levels of cytoplasmic and nuclear Rad51 were quantified as described under “Experimental Procedures.” GAPDH, glyceraldehyde-3- phosphate dehydrogenase.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques: Transfection, Western Blot