Anti hk1

Cells were lysed using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) for protein extraction. Protein concentrations were quantified using a Pierce^™ BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. A 10-μg protein was mixed with 5X SDS-PAGE loading buffer (Biosesang, Gyeonggi, Korea) and boiled at 95°C for 10 min. Boiled protein samples were separated on 10% SDS-PAGE gel and were transferred to a polyvinylidene difluoride membrane (PVDF; Sigma). Blocking of the PVDF membrane was performed using 5% skimmed milk for 1 h at room temperature. The membrane was washed with TBS-T (Tris-buffered saline with 0.1% Tween 20) three times and then incubated overnight with the primary antibody diluted in 3% bovine serum albumin (BSA) at 4°C. After the overnight incubation, the PVDF membranes were incubated with secondary anti-mouse or anti-rabbit antibodies (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 h. The blots were detected using the ECL system (Pierce, Rockford, IL, USA). The primary antibodies used were anti-hypoxia-inducible factor (HIF) -1α (1:200), anti-GAPDH (1:1000), anti-HK1 (1:1000), anti-HK2 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-PFKFB3 (1:1000), and anti-PFKFB4 (1:1000, Invitrogen).

Western blotting analysis was performed using a standard protocol. Antibodies used in this study include anti-HK1 and anti-HK2 from Santa Cruz Biotechnology and anti-PKM2 from Proteintech. Immunoreactive proteins were visualized using a chemiluminescent immunodetection system (ChemiDoc XRS). ImageJ was employed to analyze the grayscale values using anti-actin (byotiome) as housekeeping control.