Fig. 5. PI 3-kinase is involved in chemotactic-peptide- but not in colchicine-induced development of polarity. (A) Effect of wortmannin on chemotactic-peptide- and colchicine-induced polarity. Neutrophils were preincubated for 10 minutes with DMSO or with 25, 50 or 100 nM wortmannin (WT) at 37°C. The incubation was continued for another 30 minutes in medium (control) or with 10 µM colchicine (colch.) or with 1 nM fNLPNTL (CP). Cells were fixed with glutaraldehyde and the percentage of polarized cells assessed using Nomarski optics for 100 cells per sample. Mean±s.e.m. of five experiments. (B) Akt phosphorylation in response to chemotactic peptide and colchicine. Stimulation with chemotactic peptide: neutrophils were preincubated for 10 minutes with DMSO or with 25, 50 or 100 nM wortmannin at 37°C. The incubation was continued for another 5 minutes in the absence or presence of 1 nM fNLPNTL. Stimulation with colchicine: neutrophils were either incubated for 40 minutes at 37°C in medium or were exposed, after preincubation in medium for 10-35 minutes, for the times indicated to 10 µM colchicine (total incubation time: 40 minutes). Cells were precipitated with TCA and subjected to immunoblotting in order to visualize phosphorylated Akt (phospho-Akt). The blot was then stripped and reprobed with an antibody that reacts with Akt independently of its activation state. Immunoblots representative of three experiments are shown.