Anti pchk1 s317

Cells were treated as indicated, washed with cold PBS, and lysed in ice-cold cytoskeletal (CSK) buffer (10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM ATP, 1 mM Na3VO4, 10 mM NaF and 0.1% Triton X-100) freshly supplemented with protease and phosphatase inhibitors (Roche). For some experiments cells were fractionated as described previously [49 ]. Protein concentrations were determined and equalized, 5x SDS-PAGE sample buffer was added to each, and they were heated to 95°C for 10 minutes. Denatured proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The following antibodies were utilized: anti-BACH1 (FANCJ) (Sigma, B1310), anti-FANCD2 (Santa Cruz Biotechnology, sc-20022), anti-FANCC (Santa Cruz Biotechnology, sc-18110), anti-FANCI (Bethyl, A301–254A), anti-pChk1(S317) (Cell Signaling, #2344), anti-Beta-Actin (Santa Cruz Biotechnology, sc-47778), anti-Lamin B1 (Abcam, AB16048) and anti-GAPDH (Santa Cruz Biotechnology, sc-32233). Appropriate HRP-conjugated secondary antibodies (Sigma-Aldrich) were used to detect protein bands upon exposure to film (CL-Xposure, Thermo Scientific). Films were scanned and quantitated using Image-J [50 (link)].

Antibodies used for Western blot analysis included the following: anti-C1orf55/SDE2 (epitope: a.a. 318–410; Sigma-Aldrich), anti-GFP (JL-8, Clontech), anti-HA (6E2, Cell Signaling), anti-pCHK1 (S317, Cell Signaling), anti-Actin (Cell Signaling), anti-CDT1 (Cell Signaling), anti-Flag (M2, Sigma-Aldrich), anti-Tubulin (Sigma-Aldrich), Cyclin E (H-12, Santa Cruz), anti-PCNA (PC-10, Santa Cruz), anti-Vinculin (H-300, Santa Cruz), anti-GST (B-14, Santa Cruz), Cyclin A (H-432, Santa Cruz), anti-γH2AX (JBW301, Millipore), anti-CDT2 (Bethyl), anti-pRPA (S33, Bethyl), pKAP-1 (S824, Bethyl), anti-ORC2 (BD Pharmingen), and anti-MUS81 (MTA30 2G10/3, Abcam). Mitomycin C, camptothecin, hydroxyurea, cycloheximide, aphidicolin, and Z-Leu-Leu-Leu-al (MG132) were purchased from Sigma-Aldrich. Rucaparib (AG-014699) was purchased from Selleckchem. MLN4924, ubiquitin vinyl sulfone, and ubiquitin aldehyde were purchased from Boston Biochem. Drugs were used at the concentrations indicated in the figure legends.