Anti gapdh

Protein samples were separated on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Billerica, MA) for protein bands detection. The membranes were blocked in 5% nonfat milk. Primary antibodies including anti-GAPDH (1:2000, Abmart, Shanghai, CHN), anti-BCL2 (1:500, Abcam, Cambridge, UK), anti-Caspase 9 (1:1000, Cell Signaling Techonolgy, MA, USA), anti-cleaved-Caspase 9 (1:500, Cell Signaling Techonolgy, MA, USA), anti-Caspase 3 (1:1000, Abcam, Cambridge, UK), and anti-cleaved-Caspase 3 (1:1000, Abcam, Cambridge, UK) were used for incubation overnight at 4 °C. The membranes were incubated with HRP-conjugated secondary antibody (1:3000, Jackson Immuno Research, PA, USA) at room temperature for 1 h and the protein bands were detected by Pierce Fast Western Blot Kit (Thermo Fisher Scientific, MA, USA).

Cell pellets were lysed in RIPA buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Sigma), and 1% protease inhibitors cocktail (Merck). Protein extracts were quantitated and loaded on 8% to 12% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred onto a PVDF membrane (Millipore). The membrane was incubated with primary antibody, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce). Detection was performed by using a chemiluminescent Western detection kit (Cell Signaling Technology). The antibodies used were Anti-caspase-3, Anti-caspase-9, Anti-PARP, Anti-pSTAT3 (Y705), Anti-Jak2, Anti-pJak2 (Y1007/Y1008), Anti-pSTAT3 (S727), Anti-STAT1, Anti-pSTAT1 (Y701), Anti-STAT5, and Anti-pSTAT5 (Y694) (Cell Signaling Technology), Anti-Lamin B, Anti-Cyclin D1 (Santa Cruz Biotechnology), Anti-Mcl-1, Anti-STAT3 (Proteintech), and Anti-GAPDH (Abmart) antibodies.

Cells or tumor tissue extracts were digested in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL). The proteins were quantified using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions. The total protein extracts were separated by 10% SDS‐PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies, including anti‐LC3B (Abcam, Cambridge, UK), anti‐ERK1/2 (Cell Signaling Technology CST, Danvers, MA), anti‐p‐ERK1/2(CST), anti‐mTOR (CST), anti‐p‐mTOR (CST), anti‐p85 (Abcam), anti‐p‐p85 (Abcam), anti‐Akt (CST), anti‐p‐Akt (CST), anti‐p70S6K (CST), anti‐p‐p70S6K (CST), anti‐p62 (CST), anti‐Craf (Abcam), anti‐p‐Craf (Abcam), anti‐Atg7 (CST), anti‐GAPDH and β‐actin (Abmart, China) overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies (Abcam) for 1 h at room temperature, and the protein bands were detected with a ChemiDoc^™ XRS+ and Image Lab TM software (Bio‐Rad, Hercules, CA).