Phosstop phosphatase inhibitor cocktail tablets

After dissection, tissues were kept at –80 °C till use. To prepare protein lysates, tissues were homogenized with an Ultra-Turrax T10 (VWR) in 10 volumes (w/v) of ice-cold modified RIPA buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA) supplied with Complete Proteinase Inhibitor Cocktail tablets (1873580, Sigma-Aldrich) and PhosSTOP phosphatase Inhibitor Cocktail tablets (0490683701, Sigma-Aldrich). After a further 5-min sonication step in an ultrasonic bath for shearing genomic DNA, the lysates were centrifuged at 16,200× g and 4 °C for 20 min to obtain the soluble protein.
Western blot analyses were performed as described earlier [44 (link)]. Primary antibodies were diluted in TBS-T (TBS with 0.1% Tween 20) and the respective dilution factors are summarized in Table A1. For total protein detection, membranes were incubated with SYPRO Ruby Protein Blot Stain (Thermo Fisher Scientific) according to the manufacturer’s protocol, prior to blocking. For caspase-3 analyses, a commercial sample of cell extracts treated with cytochrome c served as positive control (9663, Cell signaling). All fluorescence signals were detected and quantified using the ODYSSEY FC Imaging System with Image Studio software version 4.0 (both LI-COR Biosciences, Bad Homburg, Germany).

Cells and tissue samples were harvested in CHAPS lysis buffer (1% CHAPS detergent, 150 mM NaCl, 50 mM Tris pH7, 5 mM EDTA) supplemented with cOmplete protease inhibitor and PhosSTOP phosphatase inhibitor cocktail tablets (Sigma-Aldrich, St. Louis, MO). Tissue samples were homogenized in a mini-bead beater. Protein was quantified by using Pierce’s BCA Protein Assay Reagent Kit (ThermoFisher, Walton, MA) as per the manufacturer’s instructions. Total protein (20 μg) was heated at 95 °C for 5 minutes and separated by 4–12% SDS-polyacrylamide gel. Blots were transferred to PVDF membranes for 120 minutes at a constant voltage of 100 V in 4 °C. Membranes were blocked in 5% milk (w/v) in Tris-buffered saline Tween-20 (TBS-T) at 24 °C for 1 hour. The membranes were incubated with primary antibody in 5% milk TBS-T overnight at 4 °C. A list of antibodies used for analysis is presented in the Supplementary Material.