Figure 4. PDGF increases Rac1-GTP levels by increasing PtdIns(3,4,5)P₃ concentrations in 1321N1 astrocytoma cells.

1321N1 astrocytoma cells were pre-incubated with 100 nM wortmannin for 30 min (lanes 2 and 4), or transfected with SHIP2 (lanes 5 and 6). Cells were then treated with 50 ng/ml PDGF (lanes 3, 4 and 6) or vehicle (lanes 1, 2 and 5) for 5 min. GTP-bound Rac1 was affinity-precipitated with GST–PBD, as described in the Experimental section, and the amount of precipitated Rac1 was assessed by Western blotting (Rac1-GTP). Lysates (10 μg) were also probed with the appropriate antibodies to check the total Rac1 levels (Rac1), and SHIP2 expression (SHIP2). Results are representative of three independent experiments.