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

. CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/558783 doi: bioRxiv preprint Samples were diluted with loading buffer to final concentration: 1% SDS, 2.5% 2mercaptoethanol, 0.1% bromophenol blue, 50 mM Tris pH 6.8, 10% glycerol then boiled. Samples were run on SDS-PAGE gels, then transferred to nitrocellulose (GE Healthcare) or polyvinylidene difluoride membranes (Thermo Fisher). After transferring, samples were blocked in 5% milk in tris buffered saline with 0.1% tween 20 (TBST), then incubated overnight at 4°C in primary antibody with 2.5% milk in TBST. Then membranes were washed with TBST, incubated in horseradish peroxidase conjugated (HRP) secondary antibody for 1 hour at RT, washed with TBST, and imaged with ECL Prime (Amersham) on autoradiography film (Denville). Antibodies were: Mcm2 (BD Biosciences Cat#610700, 1:10,000), Cyclin E1 (Cell Signaling Technology #4129S, 1:2,000), p53 (Santa Cruz Biotechnology, sc-126, 1:2,000), p21 (Cell Signaling Technology, #2947S, 1:6,000) Cdc6 (Santa Cruz Biotechnology, sc-9964, 1:2,000), Cdt1 (Santa Cruz Biotechnology, sc-365305, 1:3,000), Donkey-anti-Mouse-HRP (Jackson ImmunoResearch, 1:10,000) Donkey-anti-Rabbit-HRP (Jackson ImmunoResearch, 1:10,000). Membranes were stained with Ponceau S (Sigma Aldrich) to determine equal protein loading.