Peroxidase conjugated anti mouse or anti rabbit igg

Total protein was extracted from cells with lysis buffer (Pierce, Rockford, IL, USA) and quantified with the Bradford method. A total of 50 μg of protein was separated using 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4 °C with primary antibodies against GSK-3β (1:1000, Santa Cruz Biotechnology), GAPDH (1:5000, Sigma, St. Louis, MO, USA), LC3 (1:1000, Wanleibio, China), AMPK (1:1000, Wanleibio, China), and p62 (1:1000, Wanleibio, China). After washing, the membranes were incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:200, ZSGB-BIO, China) at 37 °C for 2 h. The protein bands were visualized using ECL (Pierce, Rockford, IL, USA), and images were captured using a bio-imaging system (DNR bio-imaging systems, Jerusalem, Israel).

Total protein was extracted using lysis buffer (Beyotime Biosciences) containing PMSF (Beyotime Biosciences) and phosphatase inhibitor cocktail (Biotool, Shanghai, China) and then quantified according to the Bradford method. Total protein samples (60 μg) were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore) and incubated in TBST with 5% skim milk (BD Biosciences) at 37°C for 2 h and then different primary antibodies overnight at 4°C. The membranes were washed three times with TBST for 10 min each time and incubated with peroxidase‐conjugated antimouse or anti‐rabbit IgG (1:2000; ZSGB‐BIO) at 37°C for 2 h. Protein bands were visualized with a bio‐imaging system (DNR), and relative protein expression was determined using GAPDH or beta‐actin as a loading control. Thrombopoietin (ab196026) antibodies were purchased from Abcam. Cyclin E1 (#4129), cyclin E2 (#4132), RhoA (#2117), RhoC (#3430), MYC (#2278), EGFR (#4267), P‐EGFR (Tyr1068; #3777), P‐mTOR (Ser2448; #5536), mTOR (#2983), P‐AKT (Ser473; #4060) and AKT (#4865) antibodies were purchased from Cell Signaling Technology. CDK2 (10122‐1‐AP), P27 (25614‐1‐AP), C‐Myc (10828‐1‐AP), beta‐actin (60008‐1‐ Ig) and GAPDH (60004‐1‐Ig) antibodies were purchased from Proteintech. All experiments were repeated independently at least three times.