Increased basal activation of PI3K and Rac-GTPase in KITD814V-expressing hematopoietic progenitors. (A) 32D cells expressing either WT KIT or KITD814V were starved for 8 hours, harvested, and lysed. Equal amount of lysates were subjected to immunoprecipitation using an anti-p85α antibody followed by a PI3K lipid assay. The position of p85α-associated PI3-phosphate (PI3-P) activity is indicated to the right of the blot. (B) Cell lysates derived from the treatment described in panel A were analyzed for Rac-GTPase activation by incubating the lysates with PAK-1 p21 activated kinase binding domain-agarose and analyzing by Western blot using an anti-Rac antibody. The position of Rac-GTP (active Rac) is indicated to the right of the blot. The bottom panel shows total Rac protein in each lane.