Hif 1α

The Western-blot assays described in reference with PMID 31839825¹⁴. HPV16 E6 (1:200, Bioss Biotechnology Co., Ltd, Beijing, China), HPV16 E7 (1:200, Bioss Biotechnology), PTEN (total protein, 1:500, Wanleibio Co., Ltd, Shenyang, China), p-PTEN-S380 (serine 380 phosphorylated protein, 1:1000, Zenbio Co., Ltd., Chengdu, China), TXNIP (1:500; Proteintech Co., Ltd, Wuhan, China), HIF-1α (1:1000; Wanleibio), GLUT1 (1:500; Wanleibio), and GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA). Membranes were further incubated with peroxidase-coupled anti-mouse or anti-rabbit IgG (1:5000; Proteintech) at 37°C for 2 h. Bound proteins were visualized using the ECL Western blot kit (advansta, USA), and their densities were measured using a BioImaging systems (UVP Inc., Upland, CA, USA). Protein bands were visualized using electrochemiluminescence substrate (Pierce) and detected by using BioImaging Systems (DNR, Jerusalem, Israel). GAPDH protein levels were used as the control group to calculate relative protein levels.

Total proteins were extracted from the cultured cells with a total protein extraction kit (Keygen, Nanjing, China). The protein concentrations were detected by a BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were then separated by 6 and 8% sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS-PAGE) and blotted on polyvinylidenefluoride (PVDF) membranes. Membranes were blocked in phosphate-buffered saline/Tween-20 containing 5% non-fat milk and incubated with the following primary antibodies: DEC2 (Proteintech Group, Chicago, USA), HIF-1α (Wanleibio, China), Slug (Proteintech Group, Chicago, USA), Snail (Proteintech Group, Chicago, USA), β-actin (Sigma-Aldrich). Horse radish peroxidase–conjugated anti-rabbit or anti-mouse IgG were used as the secondary antibody (TA322704 or TA326473, ZSGB-BIO, China, 1:1000). Subsequent visualization was detected using a densitometer (GS-700, Bio-Rad Laboratories).