P1291

Total proteins were extracted from cell pellets corresponding to 2–5 ODs, as described in (Simonetti et al. 2017 (link)). Cell lysis was performed on ice using 0.3 M NaOH and 1% beta-mercaptoethanol before 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 1:3,000 peroxydase-conjugated antiperoxydase (PAP, to detect protein-A-tagged proteins) (Sigma, #P1291, RRID:AB_1079562), 1:3,000 monoclonal anti-FLAG antibody (Sigma, #F3165, RRID:AB_259529), 1:3,000 anti-CDC2 antibody (Abcam, #ab5467, RRID:AB_2074778), 1:1,000 anti-GFP antibody (Roche, #11814460001, RRID: AB_390913) and 1:5,000 goat antimouse IgG-HRP (Santa Cruz Biotechnology, #sc-2005, RRID:AB_631736). 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 or a ChemiDoc Touch Imaging System (BIORAD).

Protein extracts were made by trichloroacetic acid (TCA) extraction and analyzed by western blotting as described previously (Pai et al., 2014 (link)). TAP-tagged proteins were detected with peroxidase-antiperoxidase-soluble complex (P1291; Sigma). Cdc22-GFP was detected using antibody 1181446000 (Roche), and α-tubulin was detected with antibody T5168 (Sigma). Phos-tag Acrylamide gel was used to detect Cds1-P (Wako).

To prepare samples for Western blotting, cells were harvested by filtration, washed with 20% TCA, resuspended in 100 μl of 20% TCA, and frozen. Cell pellets were lysed with 1 ml of acid-treated glass beads in a bead beater (FastPrep-5; MP Biomedicals) at level 7.5 for 15 s, and 400 μl of 5% TCA was added before eluting from the glass beads. Lysates were frozen on dry ice and spun at 18 000 relative centrifugal force for 10 min. Pellets were resuspended in SDS-Tris solution (2% SDS and Tris 0.3M pH 10.7), boiled during 5 min and cleared by centrifugation at maximum speed for 2 min. Protein extract concentrations were measured using Pierce BCA Protein Assay solution (Thermo), and 60–90 μg of total protein were loaded with Laemmli SDS Sample Buffer (reducing) (Alfa Aesar). For western blot analysis, the following antibodies were used: anti-eIF2α (1:500; 9722, Cell Signalling), anti-Phospho-eIF2α (1:1,000; 9721, Cell Signalling), and anti-tubulin (1:10 000; sc-23948, Santa Cruz). The secondary antibodies were HRP-conjugated goat anti-mouse IgG (H+L) (1:10 000; 31430, Thermo Fisher) and anti-rabbit IgG (1:10 000; ab-6721, Abcam). TAP tag was detected with peroxide–anti-peroxide complexes (P1291; Sigma). Detection was performed using the enhanced chemiluminescence procedure (ECL kit).

Western blotting was performed using the following antibodies: peroxidase-anti-peroxidase (PAP) (P1291, Sigma, 1:2000); anti-Calmodulin binding protein (CBP) (RCBP-45A-Z, ICLab, 1:500); anti-tubulin (B-5–1–2, Sigma, 1:5000); anti-FLAG (M2, F1804, Sigma, 1:1000); anti-MYC (9E10, Agro-Bio LC; 9E11, ab56, Abcam, 1:1000; or rabbit polyclonal ab9106, Abcam, 1:1000); anti-V5 (SV5-Pk1, AbD Serotec, 1:1000); anti-HA (16B12, Ozyme; rabbit polyclonal, ab9110, Abcam, 1:1000). Protein concentrations were measured by the Bradford method and used to load equal amounts of proteins across samples. Quantification of signal intensity was performed using staining, film exposure or digital acquisition that were within the linear range of detection, as verified by loading serial dilutions of one sample, and analysed using ImageJ.

Yeast cells grown in log phase were subjected to spheroplasting by supplementing the growth medium with 0.6 M sorbitol, 25 mM Tris-HCl (pH 7.5), 10 mM DTT, and purified lytic β-1,3-glucanase. Spheroplasts were washed with a buffer containing 0.4 M sorbitol, 20 mM PIPES–KOH (pH 6.6), 150 mM potassium acetate, 2 mM magnesium acetate, 1× protease inhibitors (Sigma), and lysed by a 5-min incubation on ice in an extraction buffer containing 20 mM PIPES–KOH (pH 6.6), 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM sodium fluoride, 0.5 mM Na₃VO₄, 1× protease inhibitors (Sigma) and 1% Triton X-100. Chromatin sedimentation was carried out with 15-min centrifugation at 16,000 × g on a sucrose cushion (the extraction buffer supplemented with 30% sucrose). The pellets were washed and resuspended with the extraction buffer. Equal amounts of the whole-cell extract, non-chromatin, and chromatin fractions were analyzed with western blot with the anti-Pgk1 (22C5D8, Invitrogen, 1:8000 diluted), anti-histone H3 (ab46765, Abcam, 1:2000 diluted), and anti-TAP (P1291, Sigma-Aldrich, 1:12,000 diluted) antibodies.