Glut1

Total protein from cells were extracted in lysis buffer and quantified using the Bradford method. Sixty micrograms of protein were separated by SDS-PAGE, the protein was transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After the membrane was blocked with 5% skimmed milk, the following primary antibodies were applied and incubated separately overnight at 4°C: HPV16 E6 (1:200, Bioss Biotechnology Co., Ltd, Beijing, China), HPV16 E7 (1:200, Bioss Biotechnology Co., Ltd, Beijing, China), RRAD (1:1000; Abcam, Boston, MA, USA), p65 (total protein, 1:1000; Cell Signaling, USA), p-p65 (serine 536 Phosphorylated protein, 1:500; Wanleibio, China), HIF-1α (1:1000; Cell Signaling, USA), GLUT1 (1:500; Wanleibio, China) and GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA). The membranes were further incubated with peroxidase-coupled anti-mouse or anti-rabbit IgG (Cell Signaling Technology, Beverly, MA) at 37˚C for 2 hours. The bound proteins were visualized by using ECL (Pierce, Rockford, IL) and the densities were measured by using Bio Imaging System.

Protein was extracted from cells and tissues using ice‐cold RIPA buffer as previously described.²⁰ Equivalent amounts of protein determined by Bradford method and separated by 10% SDS‐PAGE. The protein bands were electrotransferred to nitrocellulose (NC) membrane (PALL), blocked with 5% skim milk (BD) and subsequently incubated with the primary antibodies CRKL (1:2000, Santa Cruz Biotechnology), PI3K (1:500, Sanying), p‐Akt (1:800, pThr308/Ser473, Cell Signaling), GLUT1 (Glucose transporters1, 1:500, Wanlei Biotechnology), HKII (Hexokinase II, 1:500, Wanlei Biotechnology), GSK3β (Glycogen syntheses kinase 3β, 1:500, Wanlei Biotechnology), ACTB (1:5000, TransGen Biotech) and GAPDH (1:5000, Sanying) at 4°C overnight. After incubation with the secondary antibody at RT for 3 hours, protein bands were observed by ECL and quantified using the Bio‐Rad ChemiDoc™ MP system (Bio‐Rad).