Studies in granulocytic cells attaching to fibrinogen (Fg)-coated dishes on stimulation with fMLP.
(A) Day-course study of β2 integrin induction in HL-60 cells in the presence of DMSO. HL-60 cells were treated with 1.25% DMSO for the indicated days, and whole cell lysates were subjected to immunoblotting analysis with anti-β2 integrin polyclonal antibody. In each lane, the equivalent amounts of protein (8.5 μg) from whole cell lysates were loaded. The positions of molecular markers are shown on the left in kilodaltons. Arrow indicates the position of a β2 integrin protein band. (B-C) Pyk2 and Syk are tyrosine phosphorylated in relation to attachment to Fg. Granulocytic cells were incubated for the indicated times with 1 μmol/L fMLP on Fg-coated dishes (attached/detached) or in suspension in polypropylene tubes (suspension). Lysates from the equivalent number of cells (5 × 106 cells) were immunoprecipitated with anti-Pyk2 (B) or anti-Syk (SC-929; C) polyclonal antibody. Immunoblotting analysis was performed 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. (D-E) Tyrosine phosphorylation of Pyk2 and Syk in fMLP-stimulated, Fg-attached cells was inhibited by pretreatment of the cells with anti-β2 integrin antibody. Granulocytic cells were incubated for 30 minutes on dishes coated with Fg or bovine serum albumin (BSA) in the presence or absence of 1 μmol/L fMLP. In blocking experiments, some aliquots of cells were pretreated with anti-β2 integrin monoclonal antibody IB4 or normal mouse IgG before stimulation with fMLP. Lysates from the equivalent number of cells (5 × 106 cells) were immunoprecipitated with anti-Pyk2 (D) or anti-Syk (SC-573; E) polyclonal antibody. Immunoblotting analysis was performed with antiphosphotyrosine monoclonal antibody 4G10. Each blot was stripped and reprobed with the indicated antibodies (D-E, lower panels). Arrows indicate the position of a Pyk2 (D, upper panel) or Syk (E, upper panel) protein band.
