Reduced c-Kit–induced migration and reduced Rac activation in p85α^–/–mast cells. (A) Mast cells from wild-type and p85α^-/-mice were generated and subjected to migration as described in Figure 1. Data are the mean ± SEM of 20 different fields from 1 of 3 independent experiments and are expressed as the average number of migrated cells per field. *P < .05 for WT versus p85α^-/-. (B) Wild-type (p85α^+/+) and p85α^-/-mast cells were starved for 18 to 24 hours and stimulated with SCF for indicated times. Equal amounts of cell lysates were subjected to a P21-activated kinase (PAK)–binding pull-down assay, which measures active, GTP-bound Rac as described in “Materials and methods.” Shown is the position of activated Rac (Rac-GTP). (C) Wild-type mast cells were starved for 18 to 24 hours and left untreated or treated with wortmannin for 1 hour at 37°C. Subsequently, cells were washed and stimulated with SCF for indicated times. Equal amounts of cells lysates were subjected to a Rac-GTP pull-down assay as described in “Materials and methods.” Top panel: the position of activated Rac (Rac-GTP). Bottom panel: total Rac protein in each lane.