Rabbit anti hk2

Cell lysates were mixed with sample buffers and protease inhibitors in RIPA buffer, then heated at 100°C for 5 min. Abbreviations are expanded upon when first used. The language used is objective, concise, and technical terminology is consistent and accurate. After electrophoresis on an 8% SDS-polyacrylamide gel, the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were sealed before probing with the following antibodies: rabbit anti-SMAD4 (1:1000; Proteintech, China), rabbit anti-GLUT1 (1:1000; Proteintech, China), rabbit anti-HK2 (1:1000; Proteintech, China), and rabbit anti-GAPDH (1:1000; Proteintech, China). The bound antibodies were detected using a horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibody (Proteintech, China) at a dilution of 1:3000 for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Bioscience, GE Healthcare, Piscataway, NJ, USA), and the membranes were exposed to X-ray film. The GAPDH antibody was used to confirm equal loading across gel lanes. The density of each protein band was then measured and quantified using ImageJ software.