β actin antibody

The medium and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Trizol, PrimeScript RT Master Mix and SYBR green PCR master mix were from Takara (Shiga, Japan). GRP78 and uPA antibodies were from Abcam (Cambridge, UK). β-actin antibody was from Bioworld Technology (Minneapolis, MN). GAPDH and GFP antibodies were from Sangon Biotech (Shanghai, China). β-catenin antibody was obtained from Abmart (Shanghai, China). FITC-, TRITC- and HRP-conjugated secondary antibodies were obtained from Invitrogen (Carlsbad, CA).

Cells incubated with 0, 0.04, 0.16, and 0.64 μM sunitinib for 24 hours were subjected to western blot analysis following a standard protocol. Total proteins were extracted from Kasumi-1, HL-60, and NB4 cells, respectively. Blots were probed with primary antibodies directed against Beclin-1 (Cell Signaling Technology, USA) and LC3B (Beyotime Biotech, China) according to manufacturer's recommendations. The internal reference, β-actin, was detected using β-actin antibody (Bioworld Technology, USA). All western blot analyses were repeated at least twice.

Gastrocnemius were harvested in lysis buffer containing 50 mmol/l Tris (pH 7.6), 150 mmol/l NaCl, 1 mmol/l EDTA, 1% NP-40, 1 mmol/l PMSF, 1 mmol/l Na₃VO₄ and 20 mmol/l NaF. The supernatant was decanted after centrifugation at 12,000×g at 4 °C for 15 min. The protein concentration was determined using bicinchoninic acid protein assay (BCA Protein Assay Kit, Pierce Thermo-Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. For immunoblotting, an equal amount of protein (60 μg) was loaded into 8% SDS-PAGE and transferred onto Immobilon-NC Transfer Membrane (Millipore, Bedford, MA, USA). The memb-ranes were blocked in PBS containing 2% BSA for 1 hr at room temperature, followed by incubation with primary antibodies overnight at 4 °C. The primary antibodies against β-catenin, phospho-β-catenin (Ser33/37/Thr41), GSK3β, phospho-GSK3β (Ser9), were obtained from Cell Signaling (Beverly, MA, USA), Wnt3a antibody was purchased from Abcam (Cambridge, UK) and β-actin antibody was obtained from Bioworld Technology (St. Louis, USA). The membranes were colorimetrically developed using fluorescent marked secondary antibody (Beyotime Institute of Biotechnology, Nantong, China). The quantification was performed through the measurement of the signal intensity using ImageJ software (National Institutes of Health, Bethesda, MD, USA).