Time-dependent increase in the level of tyrosine phosphorylation of the p85 subunit of PI3-kinase in GM-CSF–treated neutrophils. The cells were stimulated with GM-CSF (4 nmol/L) for 1, 5, 15, and 30 minutes at 37°C. At the indicated times, the cells were lysed under denaturing conditions and the immunoprecipitations were performed using agarose-conjugated antiphosphotyrosine antibodies. The p85 subunit of PI3-kinase was revealed by blotting with specific anti-p85 antibodies. The antibodies used in lane 5 were preneutralized by 10 mmol/L O-phospho-L-tyrosine. The data are representative of three experiments, which yielded similar results.