Ab5467

Cells in supplemented EMM were collected and whole-cell protein extract was prepared by vortexing acid-washed glass beads in 20% trichloroacetic acid (TCA) and washing beads with 5% TCA. Lysates were boiled for 5 min in Laemmli Sample buffer (4% SDS, 60 mM Tris-HCl, pH 6.8, 5% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) and analyzed by SDS-PAGE. Primary antibodies used were as follows: anti-phospho-H2A (Abcam ab17353; 1:1,000), anti-H2A (Cell Signaling 3636S; 1:1,000; re-probed after phospho-H2A), anti-GFP (Abcam ab291; 1:1,000), anti-myc (Abcam ab9106; 1:1,000) anti-PCNA (Santa Cruz sc-56; 1:1,000), and anti-cdc2 (Abcam ab5467; 1:1,000). After secondary antibody (Alexa Flour 488 or 647; 1:6,000) incubation, blots were developed using Amersham Typhoon biomolecular imager. Intensity of bands were quantified with ImageJ.

Anti-HA antibody (BioScource, Covance MMS-101P-200), anti-Mrc1 antibody (ABCAM, ab188269), anti-Cdc2 antibody (ab5467) and anti-Histone 2AX-S129-P antibody (ab17576). Secondary mouse-HRP (Dako, P0447), secondary rabbit-HRP (Dako, P0217).