Cyclin e h 12

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.

Cells were lysed with NETN300 buffer (1 % NP40, 300 mM NaCl, 0.1 mM EDTA, and 50 mM Tris [pH 7.5]) supplemented with protease inhibitor cocktail (Roche), resolved by SDS-PAGE gels and transferred onto PVDF membranes (Millipore), and then antibodies were detected using the enhanced chemiluminescence method (Western Lightening, Perkin Elmer). The following antibodies were used for Western blot analysis: FAAP20 (Sigma-Aldrich), FANCD2 (FI-17, Santa Cruz), PCNA (PC-10, Santa Cruz), Cyclin E (H-12, Santa Cruz), FANCA (Bethyl), FBW7 (Bethyl), FANCG [33 (link)], MCL-1 (Cell Signaling), GSK3β (Cell Signaling), HA (6E2, Cell Signaling), ubiquitin (P4D1, Cell Signaling), CUL1 (Cell Signaling), Flag (M2, Sigma-Aldrich), c-Myc (9E10, Sigma-Aldrich), β-Actin (Cell Signaling), Tubulin (Sigma-Aldrich), and ORC2 (BD Pharmingen). pS113 FAAP20 phospho-specific antibody was generated by Genscript using the GGHLEpSPARSLC peptide to immunize rabbits.