Semi-Dry Transfer Unit for WB

Cells were rinsed with cold PBS and lysed in RIPA lysis buffer (0.1% NP40, 20 mM Tris/HCl pH 8, 10% Glycerol, with fresh protease inhibitors (Roche #04693132001) and phosphatase inhibitors (Calbiochem #524629). Cell lysates were cleared by centrifugation in a microcentrifuge (15,000 rpm for 10 minutes at 4°C). Cell lysates were prepared by addition of 2x Laemmli sample buffer (4% SDS, 20% Glycerol, 120 mM Tris-Cl pH 6.8, 0.02% bromophenol blue, and 5% β-Mercaptoethanol, resolved by 4%- 20% SDS-PAGE), and analyzed by immunoblotting.