Whole-cell lysates were prepared using RIPA buffer (Sigma) containing protease inhibitor cocktail, and protein concentration was assessed by BCA colormetric protein determination (Pierce). Equal protein loads were resolved on precast 4% to 12% Bis-Tris gels under reducing conditions. Protein was transferred to nitrocellulose membranes using iBlot dry transfer method (Invitrogen), then probed with primary antibody anti-RAD51C (sc-398819 1:1,000; Santa Cruz Biotechnology), anti-RAD51D n-terminal (ab202063 1:1,000; Abcam), anti-RAD51D c-terminal (sc-398819 1:1,000; Santa Cruz Biotechnology), anti-GAPDH (#5174 1:1,000; Cell Signaling Technology), anti-tubulin (#2128 1:1,000; Cell Signaling Technology) or anti-actin (ab8229 1:1,000; Abcam) followed by peroxidase-labeled secondary antibody (sc-2020 1:5,000; Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence (SuperSignal Chemiluminescent Substrate; Thermo Scientific) or IRDye-labeled secondary antibody (LI-COR Biotechnology) according to the manufacturer's instructions. Band volume analysis was conducted using Odyssey Fc (LI-COR Biotechnology).
Kondrashova O., Nguyen M., Shield-Artin K., Tinker A.V., Teng N.N., Harrell M.I., Kuiper M.J., Ho G.Y., Barker H., Jasin M., Prakash R., Kass E.M., Sullivan M.R., Brunette G.J., Bernstein K.A., Coleman R.L., Floquet A., Friedlander M., Kichenadasse G., O'Malley D.M., Oza A., Sun J., Robillard L., Maloney L., Giordano H., Wakefield M.J., Kaufmann S.H., Simmons A.D., Harding T.C., Raponi M., McNeish I.A., Swisher E.M., Lin K.K, & Scott C.L. (2017). Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma. Cancer discovery, 7(9), 984-998.
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