Anti pckit

For SCF/c-Kit signaling analysis, the cells were serum starved for 30 min and then pretreated with 4C9 antibody at the indicated concentrations for 30 min. Next, 100 ng/mL recombinant human SCF (R&D Systems, Minneapolis, MN, USA) was added and incubated for an additional 5 min. The cells were washed twice with DPBS and lysed using RIPA buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 10 mM β-glycerophosphate, 1 mM Na3OV4, 10 mM NaF, 1 μg/mL leupeptin, 1 mM PMSF, 5 μg/mL aprotinin, and 2 mM 2-mercaptoethanol). The protein extracts were subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 1 × TBST containing 5% BSA at room temperature (RT) for 1 h and probed with the following antibodies: anti-c-Kit (R&D Systems, Minneapolis, MN, USA), anti-p-c-Kit (Tyr568/570, Tyr703, Tyr719, and Tyr823; Cell Signaling Technology, Beverly, MA, USA), anti-Erk1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-Erk1/2 (Cell Signaling Technology, Beverly, MA, USA), anti-Akt (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-Akt (Ser473; Cell Signaling Technology, Beverly, MA, USA), and anti-α-tubulin (generated in our laboratory).