Electrochemilumine scene detection system

Western blotting was employed to detect the protein level of HK2 as previously described. β-Actin (Abcam) was used as the protein-loading control. The protein complex was detected with enhanced chemiluminescence reagents. Digital images were visualized using the electrochemilumine scene detection system (Invitrogen, USA).

Cells were collected 48 h after infection. The total protein was dissociated in RIPA lysis buffer (Beyotime, Shanghai, China). The protein contents were measured using a BCA assay (Beyotime, Shanghai, China) and separated on 8% SDS-PAGE gel, and electrotransferred to PVDF membranes. PVDF membranes were blocked with 5% skim milk for 2 h in TBST and then incubated with initial antibodies at 4 °C overnight. The antibodies used were: mouse HK2 antibody (1:1,000; Abcam, Cambridge, USA) and mouse β-actin (1:10,000; Santa Cruz, California, USA). The secondary antibody was peroxidase-conjugated Affinipure goat anti-mouse IgG (H + L) (ZSGB-BIO, Beijing, China). After incubation with a secondary antibody, the membranes were mixed with chemiluminescence reagents. Digital images were visualized though the electrochemilumine scene detection system (Invitrogen, USA).