Hexokinase 2 hk2

Total protein from transfected HL-60 and KG-1 cells was isolated in RIPA lysis buffer (Beyotime) supplemented with cocktail protease inhibitor (Roche), and the protein concentrations were determined by Bradford protein assay reagent (Bio-Rad). Equal amounts of protein (20 μg) from each sample were loaded for the standard procedures of Western blot assay. β-actin on the same membrane was an internal standard to normalize protein levels. The primary antibodies including hexokinase 2 (HK2; #2867, 1:1000) and β-actin (#58169, 1:1000) were purchased from Cell Signaling Technology (CST; Danvers, Massachusetts, USA).

Total protein of the CRC cells was prepared using RIPA lysis buffer (Beyotime, Nantong, China). The protein concentration was detected using a Bradford protein assay kit (Bio-Rad). A total of 50 μg of protein was isolated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by electro-blotting-mediated transfer onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then blocked in 5% non-fat milk in TBST buffer (5 mM Tris–HCl, pH 7.4, 136 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, and then incubated with primary antibodies against bcl-2, bax, cleaved caspase-3, hexokinase 2 (HK2), glucose transporter 1 (GLUT1), p-PI3K, PI3K, p-Akt, Akt, and GAPDH (Cell Signaling, Danvers, MA, USA) overnight at 4 °C. After washing three times with TBST, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Cell Signaling) for 1 h. Finally, the immunocomplexes were visualized by chemiluminescence using an ECL kit (Amersham Biosciences, Piscataway, NJ, USA).