Enhanced chemiluminescence kit

Radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) was used to extract total protein from BxPC-3 and Capan-2 cells, and protein quantity was determined by bicinchoninic acid assay. Gel electrophoresis was performed using 10% SDS-PAGE with 25 µg per lane and the proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 2 h at room temperature, followed by incubation with primary antibodies including rabbit anti-HK2 (dilution, 1:2,000; cat. no. ab37593) and anti-GAPDH primary antibody (dilution, 1:1,000; cat. no. ab8245) (both Abcam, Cambridge, MA, USA) overnight at 4°C. The following day, membranes were washed in triplicate with PBS for 15 min, and incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (dilution, 1:1,000; cat. no. MBS435036; MyBioSource, Inc., San Diego, CA, USA) at room temperature for 2 h, followed by signal detection using an enhanced chemiluminescence kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Signals were scanned using MYECL™ Imager (Thermo Fisher Scientific, Inc.), and ImageJ v.1.48 software (National Institutes of Health, Bethesda, MD, USA) was used to normalize the relative expression level of HK2 to the endogenous GAPDH control levels.

The protein expression levels of p-MET, p-AKT, p-ERK, MET, AKT and ERK were analyzed by western blot analysis. On day 21 of the efficacy study, at 2 h following the final injection of GA, the mice were humanely sacrificed. The tumors were harvested in lysis buffer (Cell Signaling Technology, Inc.) and homogenized using Misonix Sonicator 4000 (Misonix Inc., Farmingdale, NY, USA); protein lysates were generated and protein concentrations were determined using a bicinchoninic acid assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of protein (50 µg) were then separated by SDS-PAGE on 10% gels, blotted on polyvinylidene difluoride membranes (Sigma-Aldrich) and probed with p-MET, p-AKT, p-ERK, MET, AKT and ERK rabbit polyclonal primary antibodies (dilution, 1:1,000; incubation, overnight at 4°C) and subsequently with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (#sc-2040; dilution, 1:1,000; incubation, 1 h at room temperature), and detected with an enhanced chemiluminescence kit (Sigma-Aldrich).

Total proteins were isolated from SNU-398 cells using RIPA lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.). Protein concentrations were measured using a BCA assay (Sigma-Aldrich; Merck KGaA). All protein samples were denatured at 95°C for 10 min, followed by loading 25 µg protein/lane onto 8% gels and resolving using SDS-PAGE. Then these samples were transferred to the polyvinylidene fluoride (PVDF) membranes. All membranes were blocked with PBS containing 5% non-fat milk at room temperature for 2 h. Primary rabbit antibody of GAPDH (1:1,000; cat. no. ab9485) or PTEN (1:500; cat. no. ab14993) (both Abcam) was used to incubate with the membranes at 4°C for 12 h, followed by incubation with HRP Goat Anti-Rabbit (IgG) (1:1,000; ab97051; Abcam) secondary antibody at room temperature for 2 h. Enhanced chemiluminescence kit (Sigma-Aldrich; Merck KGaA) was used to visualize the signals, which were quantified by Quantity One software version 4.6 (Bio-Rad Laboratories, Inc.).