Amersham enhanced chemiluminescence western blotting detection kit

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).

Protein concentration was determined by bicinchoninic acid protein quantification. Total protein (20 µg) was isolated from 10 paired HCC and adjacent non-malignant tissue samples using TRIzol^® buffer (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) (21 (link)) as previously described (22 (link)). Briefly, equal quantities of whole cell lysate and tissue lysate were separated by 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Pall Life Sciences, Port Washington, NY, USA) and blocked in 5% milk powder. The membranes were washed three times with PBS and incubated with primary mouse monoclonal antibodies against human IGFBP-3 (dilution, 1:200; catalog no. sc-9028; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Subsequent to washing three times with PBS, blots were incubated with secondary rabbit anti-mouse antibody (catalog no. F0232; Envision; Dako; Agilent Technologies, Inc.) at a dilution of 1:5,000 for 2 h at room temperature. Immunoreactivity was then detected using an Amersham enhanced chemiluminescence western blotting detection kit (GE Healthcare Life Sciences, Uppsala, Sweden) and analyzed using Image lab Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were conducted according to the manufacturer's protocol where applicable.