Dose dependence and time-course studies of tyrosine phosphorylation of Pyk2 on stimulation with
N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in granulocytic cells. HL-60 cells were treated with 1.25% DMSO for 4 days, and the differentiated (granulocytic) cells were then incubated in suspension in polypropylene tubes for 1 minute with the indicated concentrations of fMLP (A) or for the indicated times with 1 μmol/L fMLP (B). Lysates from the equivalent number of cells (5 × 106 cells) were immunoprecipitated with anti-Pyk2 polyclonal antibody (A) or with anti-Pyk2 polyclonal antibody, normal goat IgG, or protein A alone (B). In the experiment shown in Figure 2B, an aliquot of cells was pretreated with pertussis toxin (PT) before stimulation with fMLP. Immunoblotting analysis was performed with antiphosphotyrosine monoclonal antibody 4G10. Each blot was stripped and reprobed with anti-Pyk2 polyclonal antibody (A-B, lower panels). Arrows indicate the position of a Pyk2 protein band (A-B, upper panels).
