Anti fancd2 nb100 182

For immunoblotting analysis, cell suspensions were washed in ice-cold PBS and lysed in 50 mM Tris.Cl pH 7.4, 1.0% v/v NP-40, 0.25% w/v Deoxycholic acid, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM Na₃O₄V, 1 mM NaF, plus protease inhibitors cocktail (Roche). The cellular fractionation method is described in the Supplemental Experimental Procedures. Proteins were resolved on NuPAGE 3–8% w/v Tris-Acetate or 4–12% w/v Bis-Tris gels (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes. The following antibodies were used: rabbit polyclonal anti-53BP1 (sc-22760; Santa Cruz Biotechnology), anti-FANCA (ABP6201; Cascade), anti-FANCD2 (NB100–182; Novus Biologicals), anti-FANCI (Dr. Patrick Sung, Yale University and A301-254A; Bethyl Laboratories), anti-FANCM (a kind gift from Dr. Ruhikanta Meetei, Cincinnati Children’s Hospital), anti-FANCM (3821; Fanconi Anemia Research Fund), anti-H2A (07–146; Millipore), and anti-PTEN (9559;Cell Signaling), and mouse monoclonal anti-FANCM (CP3.2, CV11.1, and CV5.1), anti-γH2AX (05–636;Millipore), anti-PTEN (6H2.1;Cascade), anti-RAD51 (sc-8349; Santa Cruz), and anti-α-tubulin (MS-581-PO; Lab Vision).