Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

Jacob Peter Matson; Amy M. House; Gavin D. Grant; Huaitong Wu; Joanna Perez; Jeanette Gowen Cook

To prepare chromatin fractions for immunoblot, cells were harvested with trypsin and frozen in dry ice, then lysed in cold CSK with 0.5% triton x-100, 1 mM ATP, 5 mM CaCl2 and protease and phosphatase inhibitors (complete CSK) on ice for 20 minutes. Then a Bradford assay for equal loading was done on the lysate, a small aliquot removed from each sample as total lysate, and complete CSK was added to each sample, mixed, and centrifuged for 5 min, 1,000 x g. The supernatant was removed, pellet was washed again with complete CSK, incubated for 10 minutes on ice, and then centrifuged for 5 min, 1,000 x g. The supernatant was removed, and DNA loaded proteins were released by incubation with S7 nuclease (Sigma Aldrich) in complete CSK at room temperature (RT) for 10 minutes. Samples were centrifuged again, keeping the supernatant as the chromatin fraction.