Tyrosine phosphorylation of KIT and the association of p85^PI3-K with KIT in Ba/F3 cells stably expressing various Val814 mutants.

(A) KIT was immunoprecipitated with ACK2 mAb from lysates of the indicated cells before and after stimulation with rmSCF (100 ng/mL) for 10 minutes at 37°C. Immunoprecipitates were divided into 2 aliquots, separated by SDS-PAGE, and subjected to immunoblotting with antiphosphotyrosine mAb (α–P-Tyr, top panel) or with a rabbit polyclonal antibody against the whole murine KIT (α–c-kit, bottom panel). Mobilities of the mature (145 kDa) and immature (125 kDa) forms of KIT are indicated at the right. (B) KIT or p85^PI3-K was immunoprecipitated with ACK2 mAb (top panel) or a rabbit polyclonal antibody against N-terminal SH2 domain of p85^PI3-K(α-p85^PI3-K, bottom panel) from the same lysates described in the legend to Figure 4A. Immunoprecipitates were separated and subjected to immunoblotting with α-p85^PI3-K. The position of p85^PI3-K is indicated at the right. In all experiments represented by Figure4, cells transfected with pEF-BOS vector alone (Vector) were used as a negative control. Similar results were obtained from 3 independent experiments. Y represents tyrosine (Tyr); F, phenylalanine (Phe).