Protease inhibitor and phosstop phosphatase inhibitor cocktail tablets

Protein extracts were prepared using RIPA buffer supplemented with Protease Inhibitor and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche). Histones were isolated using Histone Extraction Kit (Abcam) according to the manufacturer’s instructions. Protein extracts were PAGE-separated, electrotransferred to PVDF membranes (Millipore), blocked in 5% bovine serum or nonfat milk, and then immunoblotted with primary and appropriate secondary antibodies (Table S4). Signals were detected and quantified as described elsewhere [47 (link)].

Cells were centrifuged (300 × g, 5 min, 4 °C), washed with PBS and suspended in RIPA buffer supplemented with Protease Inhibitor and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche) according to the manufacturer’s protocol. The protein extracts were quantified using Pierce BCA Protein Assay Kit (ThermoFisher). Total protein extracts (20 μg–40 μg) were mixed with 4× Laemmli sample buffer supplemented with 2-mercaptoethanol and boiled at 95 °C for 5 min. The prepared samples were SDS-PAGE-separated, electrotransferred to PVDF membranes (Millipore), blocked in 5% bovine serum or non-fat milk, and then immunoblotted with primary and appropriate secondary antibodies (Table S4). Signals were detected and quantified as described previously [30 (link)]. Densitometry analysis was performed using Image Studio Lite Quantification Software (Licor). The results obtained for the investigated proteins were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, loading control).