Laemmli s sds sample buffer

Each frozen tissue/cell pellet was homogenized in 10× volume of RIPA lysis buffer (10 mM Tris-Cl [pH 7.2], 150 mM NaCl, 1 mM EDTA [pH 8]) with 1% Triton X-10/0.1% deoxycholate, 0.1% SDS (all from Sigma-Aldrich), and protease and phosphatase inhibitor mixture (Roche). Samples were then diluted in Laemmli’s SDS sample buffer (Bio-Rad). Proteins were separated by electrophoresis on 10% polyacrylamide gels according to the TGX Stain-Free FastCast Acrylamide kit protocol (Bio-Rad), and they were transferred onto Trans-Blot nitrocellulose membranes (Bio-Rad) according to the Trans-Blot Turbo Transfer System kit protocol (Bio-Rad). Primary antibodies were diluted in 3% BSA (Sigma-Aldrich) or 5% nonfat dry milk in TBS-T, and the membranes were incubated overnight at 4°C (see Supplemental Methods). The primary antibody was removed, and the blots were washed in TBS-T and then incubated for 1 hour in HRP-conjugated secondary antibodies (Amersham). Reactive proteins were visualized using a Clarity Western ECL substrate kit (Bio-Rad), and exposure was performed using UVItec (Cambridge MINI HD). Images were acquired by NineAlliance software.