Protein Extraction and Analysis Protocol

Lysates for western blotting and immunoprecipitation were prepared using the normal lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100) containing protease inhibitor cocktail (p8340, Sigma) and 0.2 mM sodium orthovanadate. Immunoprecipitation was performed using protein-A/G agarose beads (Invitrogen), as previously described [27 (link)]. SDS-PAGE electrophoresis and western blotting were performed using the NuPAGE SDS PAGE Gel System and NuPAGE Bis Tris Precast Gels (4–12%) (Life Technologies). Western Lightning PLUS Enhanced Chemiluminescent Substrate (PerkinElmer) was for imaging western blots on the Vilber Lourmat Fusion chemiluminescent imaging system. Quantitative western blotting was performed using multistrip western blotting [28 (link)]. The Human Phospho-Kinase Antibody Array was obtained from R&D Systems (MN, USA) and used according to manufacturer’s instructions.