Preparation and Immunoblotting Protocol

293T whole cell extracts and mouse liver lysates were prepared using Lysis buffer containing 1%TX-100 as previously described (Lamia et al., 2004 (link)). MEF cell extracts were prepared from RIPA buffer containing 1% TX-100, 147 mM NaCl, 12 mM sodium deoxycolate, 0.1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA, 50 μM PMSF, phosphatase inhibitors (cat #P5266 and cat #P0044; Sigma, St. Louis, MO) and protease inhibitor (cat #11697498001; Roche, Switzerland). For ubiquitination experiments, iodoacetamide was added to the buffer to a final concentration of 5 mM (Fisher AC122270050).
Antibodies were anti-Flag M2 agarose beads, anti-Flag polyclonal, anti-v5 polyclonal, anti-Lamin A, anti-aTubulin, and anti-βactin from Sigma, anti-Hausp and anti-V5 from Bethyl Labs (Montgomery, TX, cat #A300-033A and cat #A190-120A), anti-53BP1 from Novus Biologicals (Littleton, CO, NB100-304), Cry1-CT and Cry2-CT as described (Lamia et al., 2011 (link)), anti-p21 from Santa Cruz Biotechnologies (Dallas, TX, cat #sc-6246), anti-p53 as previously described (Pasini et al., 2004 (link)), and anti-Ubiquitin, anti-phospho-P53 (S15), and anti-phosphoATM/ATR substrate (phospho-SQ/TQ) from Cell Signaling Technology (Danvers, MA). Anti-Cry1-phosphoS588 antibody was affinity purified from rabbit antisera raised against a phospho-S588 containing peptide.