fMLP- and TPA-induced Rap1 activation is independent of PKC. (A) fMLP-induced Rap1 activation is not inhibited by PKC inhibitors. Neutrophils were preincubated for 5 minutes with 200 nmol/L staurosporine or 10 minutes with 5 μmol/L GF109203X. Neutrophils were then stimulated with 1 μmol/L fMLP for 10 seconds and 5 minutes. As a control, untreated neutrophils of the same donor were used. (B) TPA-induced Rap1 activity is not inhibited by a PKC inhibitor. Neutrophils were stimulated with 100 ng/mL TPA for 5 and 10 minutes after preincubation for 5 minutes with 200 nmol/L staurosporine or buffer. Oxygen consumption was measured with a Clark oxygen electrode to measure the functionality of staurosporine treatment. Representative examples of at least three independent experiments are shown. (C) Inhibition of PLCβ does not influence Rap1 activity. Neutrophils were preincubated with 1 μmol/L U73122 (PLCβ inhibitor) for 3 minutes and subsequently stimulated with 1 μmol/L fMLP. (D) Rap1 activity is not inhibited by PI-3 kinase inhibitors. Neutrophils were preincubated with the PI-3 kinase inhibitors LY294002 (10 μmol/L) or wortmannin (100 nmol/L) for 5 minutes. Samples were taken after preincubation and 10 seconds and 5 minutes after 1 μmol/L fMLP stimulation. As a control, neutrophils of the same donor without preincubation were used. Representative examples of at least three independent experiments are shown.