Involvement of SFK in SCF-induced migration and Ca²⁺influx. (

A) SCF-induced activation of Lyn. 293T cells were transiently cotransfected with Lyn and c-Kit constructs. After 18 hours of starving from serum, cells were stimulated with SCF (100 ng/mL) for 10 minutes and lysed. Total cell lysates were blotted with antiphospho Src antibody, which detects activated forms of SFK. Stripped membrane was reblotted with anti-Lyn antibody to prove the same loading. (B) Expression of Csk constructs. BAF3/KIT cells were stably transfected with WT-Csk, kinase dead Csk (Csk(−)) and constitutive active Csk (membrane-associated Csk; mCsk). The amounts of Csk proteins in total cell lysates were detected by blotting with anti-Csk. (C) Inhibition of Ca²⁺ influx by Csk. SCF-induced Ca²⁺ influx was measured in 293T cells transiently cotransfected with c-kit and Csk constructs BAF3/KIT cells stably transfected with Csk constructs. (D) Inhibition of cell migration by Csk. Cell migration toward 30 ng/mL SCF was analyzed in BAF3/KIT-expressing Csk constructs. Data are shown the same as Figure 3C. The asterisk represents a significant difference from a control (Mock) value (P < .01). Similar results were obtained from 3 independent experiments.