Protein Extraction and Immunoblotting

Total proteins were extracted from cell pellets corresponding to 2 to 5 ODs, as previously described^{45 (link)}. Briefly, cell lysis was performed on ice using 0.3 M NaOH and 1% beta-mercaptoethanol prior to protein precipitation with trichloroacetic acid (TCA) (7% final). Following full speed centrifugation, pellets were resuspended in HU loading buffer and heat-denaturated at 70˚C. Soluble fractions were recovered, and samples were analyzed by standard immunoblotting procedures using 1:3000 peroxydase-conjugated antiperoxydase (PAP, to detect protein-A-tagged proteins) (Sigma, #P1291, RRID:AB_1079562), 1:3000 monoclonal anti-FLAG antibody (Sigma, #F3165, RRID:AB_259529), 1:3000 anti-CDC2 antibody (Abcam, #ab5467, RRID:AB_2074778), 1:1000 anti-GFP antibody (Roche, # 11814460001, RRID: AB_390913), 1:500 anti-CBP antibody (Millipore, #07-482, RRID:AB_310653), 1:5000 goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, #sc-2005, RRID:AB_631736) and 1:5000 goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, #sc-2004, RRID:AB_631746). Detection was done with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific, #34080), ECL Select reagent (GE Healthcare, #RPN2235), and a Vilber Lourmat Fusion Fx7 imager.