In vitro kinase assays of anti-β2 integrin, anti-Pyk2, and anti-Syk immunoprecipitates from lysates of fMLP-stimulated granulocytic cells.
(A) Granulocytic cells were incubated for 30 minutes with 1 μmol/L fMLP on Fg-coated dishes (attached) or in suspension in polypropylene tubes (suspension). Anti-β2 integrin, anti-Pyk2, and anti-Syk (SC-573) immunoprecipitates from lysates of the equivalent number of cells (5 × 106 cells) were mixed as indicated and subjected to in vitro kinase assays in the presence or absence of adenosine triphosphate (ATP) and immunoblotting with antiphosphotyrosine monoclonal antibody 4G10. Each blot was stripped and reprobed with anti-β2 integrin polyclonal antibody. The positions of molecular markers are shown on the left in kilodaltons. Arrows indicate the position of a Pyk2, Syk (upper panel), or β2 integrin (lower panel) protein band. (B-C) Granulocytic cells were incubated for 30 minutes with 1 μmol/L fMLP on Fg-coated dishes (attached) or in suspension in polypropylene tubes (suspension). Anti-Pyk2 (B) and anti-Syk (SC-929; C) immunoprecipitates from lysates of the equivalent number of cells (5 × 106 cells) were subjected to in vitro kinase assays in the presence or absence of ATP and immunoblotting with antiphosphotyrosine monoclonal antibody 4G10. Each blot was stripped and reprobed with the indicated antibodies (B-C, lower panels). Arrows indicate the position of a Pyk2 (B, upper panel) or Syk (C, upper panel) protein band.
