Anti par

The indicated cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% NP-40, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, and 0.1 mg/mL leupeptin/aprotinin) on ice for 30 min, centrifugated at 15,000×g for 30 min, and the supernatants were collected for Western blotting. The primary antibodies were incubated with the membrane overnight at 4 °C. Horseradish peroxidase-conjugated goat anti-rabbit-mouse or rabbit IgG secondary antibodies (Beyotime, Haimen, China) were incubated with the nitrocellulose blotting membranes (GE Healthcare Life science, Boston, Massachusetts) for 1 h at room temperature in the next morning. Images were obtained using a chemiluminescence (ECL) detection system (ProteinSimple, San Jose, CA). Quantified band intensities were normalized using β-actin as housekeeping protein. Blots were scanned with a Tanon imaging system (5200, Shanghai, China). The primary antibodies used included anti-STAT3, anti-ERK1/2, anti-p-STAT3, anti-p-ERK1/2, anti- PI3Kγ, anti-HRAS, anti-SAP18, anti-JAK, anti-p-JAK, anti-SHP2, anti-p-SHP2, anti-PARP1, anti-PAR, and anti-β-actin, which were all purchased from Santa Cruz Biotechnology and Cell Signaling Technology.