“…21,90,91 Immunoblot analysis Cell and tissue samples were lysed in lysis buffer. 92 Samples were dissolved in 50 mM Tris-HCl, pH 7.4, 250 mM sucrose (Bioshop, SUC507), 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 (Sigma, T8787), 0.1 mM benzamidine (Sigma, B6506), 1 mM DTT, 0.5 mM PMSF (Sigma, P7626), 50 mM NaF, protease inhibitor cocktail (Calbiochem, 535140), and phosphatase inhibitor cocktail (Sigma, P5726). Lysates were resolved on SDS-polyacrylamide gels and blotted onto PVDF membranes (Millipore, IPVH00010) for 35 min at 20 V in transfer buffer (25 mM Tris base, pH 7.4, 192 mM glycine, 10% methanol).…”