Detection of PDGFR phosphorylation by immunoprecipitation/Western blotting

To measure levels of endogenous phosphorylation of PDGFR-β, cells were harvested in lysis buffer (50 mM Tris-HCl pH 7.5, 1% Tween-20, 200 mM NaCl, 0.2% NP-40, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, and protease inhibitors). Anti-PDGFR-β antibody was used to immunoprecipitate the receptor protein from a whole cell lysate containing 2 mg of cellular proteins. Immunoprecipitates were washed, subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an anti-phosphor-tyrosine antibody to study the phosphorylation status of PDGFR-β.