Fig. 4.

The role of PI3K in synergistic chemotactic responses. (A) Sensitized BMMCs were preincubated with or without wortmannin (100 nM), in the upper chamber and placed in 600 μl HEPES buffer containing 0.5% BSA and wortmannin for 30 minutes. The upper chambers were then replaced in the lower chambers containing the indicated agonists. (B) Sensitized BMMCs were preincubated with indicated inhibitors for 20 minutes and then stimulated with the indicated agonists for 5 minutes. Following electrophoresis and membrane transfer, proteins were probed using anti-phospho-AKT (Ser473-P). To normalize protein loading, membranes were stripped and probed for β-actin, or alternatively identically loaded samples were probed for β-actin. The data shown are from three separate experiments, each repeated at least three times, with identical results, on separate cell preparations. (C) Sensitized BMMCs were preincubated with or without AS 252424 (3 μM) or IC 87114 (3 μM) in the upper chambers and placed in 600 μl HEPES buffer containing 0.5% BSA and indicated inhibitors for 30 minutes, and then upper chambers were replaced in the lower chamber containing the indicated agonists. Results in A and C are means ± s.e. of three separate experiments. *P<0.05 for comparison with the same stimulation in control group by Student's t-test.