P c kit

Cells were harvested and lysed using RIPA buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 2.5 mM NaF, 5 mM Na₃VO₄, and protease inhibitor cocktail tablet (Roche, # 11–878-580–001). Proteins were separated and transferred to an NC membrane. The membranes were blocked using 5% skim milk in TBS-T buffer. The p-c-KIT (Tyr703, # 3073), p-AKT (Ser473, # 9271), p-ERK (Thr202/Tyr204, # 8544), ERK (# 4695), p-PLCγ (Tyr783, # 14008), p-STAT3 (Tyr705, # 4074), and PARP (# 9542) antibodies were obtained from Cell Signaling Technologies. The β-actin (# sc-47778) and STAT3 (# sc-482) antibodies were obtained from Santa Cruz Biotechnology. AKT (A18120) antibody was purchased from ABclonal. Each primary antibody was incubated overnight at 4 °C. The secondary antibody (GenDEPOT) was incubated for 1 h at room temperature. Proteins were detected using ECL substrate.

Western blot assay was performed as previously described.31, 32 Total cell protein was isolated using NP40 lysates in the presence of inhibitors of proteases and phosphatases. The proteins were separated by SDS‐PAGE and transferred to PVDF membranes. Blots were incubated overnight with primary antibodies at 4°C. Then, blots were incubated with the corresponding secondary antibodies conjugated with HRP. Immunoreactive bands were examined with the ECL reagent from Thermo Scientific. The primary antibodies are list as followed: BRD4 (#13440), cleaved caspase 3 (#9661), cleaved caspase 9 (#9509), LC3 I/II (#4108), p‐PI3K (#4228), PI3K (#4255), p‐AKT (#4060), AKT (#9272), p‐mTOR (#2976), mTOR (#2972), p‐cKIT (#3391), cKIT (#3074, Cell Signaling Technology) and β‐actin (#5441, Sigma).