Igf 1

Lysates were resolved by electrophoresis, transferred to a polyvinylidene difluoride membrane (Millipore Corporation), and probed with antibodies against GAPDH (Santa Cruz), VEGF (Santa Cruz), SMAD2 (CST), IGF1 (Santa Cruz), SMAD4 (CST), JAK2 (CST), HGF (Abcam), Phospho-STAT3 (CST), Phospho-c-Jun (CST).

Whole cell extracts were prepared by the lysis of cells in buffer (60 mM Tris-HCl, pH 6.8, 5% glycerol, 2% SDS) on ice. Proteins were quantified using Pierce BCA (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cellular proteins (50 µg) were solubilized in sample buffer (4% SDS, 0.25 m sucrose, 30 mm , 0.075% bromophenol blue and 0.01 m EDTA-Na₂) and denatured at for 5 min. The proteins were separated by SDS-PAGE using 4% stacking and 12% separating gels and then electro-transferred onto polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked in 0.05 M Tris-buffered saline with 0.5% triton X-100 (TBS-T, pH 7.4) containing 5% skim milk at room temperature for 2 h and then incubated in 3% skim milk or bovine serum albumin at overnight with primary antibody. The primary antibodies used were IGF-1 (1:1000 dilution; Santa Cruz, CA, USA) p-AKT (1:500 dilution; CST, USA) p-ERK1/2 (1:1000 dilution; Santa Cruz, CA, USA) GAPDH (1:1000 dilution; Santa Cruz, CA, USA). After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 for anti-rabbit-IgG or anti-mouse-IgG) for 1 h at room temperature. Proteins were detected by the Amersham enhanced chemiluminescence Western blotting detection kit (GE Healthcare, Piscataway, NJ).