Effects of phorbol esters on the tyrosine phosphorylation of p85 and on the pattern of tyrosine phosphorylation in human neutrophils. Neutrophils were simultaneously treated with BSA (0.01%), GM-CSF (4 nmol/L), and/or phorbol esters (90 nmol/L) for 15 minutes at 37°C. (A) Following denaturing lysis, immunoprecipitation was performed with agarose-conjugated antiphosphotyrosine antibodies and p85 was revealed by blotting with anti-p85 antibodies. (B) Cells were lysed in boiling sample buffer and blotting was conducted using antiphosphotyrosine antibodies. The samples in (A) and (B) were derived from the same experiment, which was repeated four times with similar results.