Anti hif 1α

Stable overexpression cells were grown up to 90% confluency in 6 well plates and lysed in RIPA buffer with 1 mM DTT, 1 mM benzamidine, 1 mM PMSF and 1× protease inhibitor cocktail. Cell resuspensions were incubated for 15 min on ice, followed by short sonication. The supernatants of lysates were collected after centrifugation at 10,000 rpm for 10 min at 4 °C. The samples were heated with 1× SDS loading buffer at 98 °C for 5 to 10 min. Following 10% or 15% SDS-PAGE and wet blot transfer, the blots were probed with one of the following primary antibodies in TBST: monoclonal anti-FLAG^® M2 antibody (Merck KGaA, Cat. No. F1804, Darmstadt, Germany), mTOR (phosphor-S2448) polyclonal antibody (Bioworld, Cat. No. BS4706, Nanjing, China), mTOR (S2442) polyclonal antibody (Bioworld, Cat. No. BS3611, Nanjing, China), anti-HIF-1α (Sangon, Cat. No. D162108, Shanghai, China); anti-LC3 (abcam, Cat. No. ab51520, Cambridge, UK); anti-p62 (abcam, Cat. No. ab56416, Cambridge, UK); anti-GFP (Proteintech, Cat. No. 66002-1-Ig, Wuhan, China), anti-GAPDH (Proteintech, Cat. No.60004-1-Ig, Wuhan, China).

The tissues and cultured cells were lysed and the protein was extracted using RIPA buffer (Solarbio, Beijing, China). The protein was quantified using a BCA kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Protein lysates were separated using 10% SDS-PAGE gel electrophoresis and transferred to PVDF membrane (Bio-Rad, Richmond, CA, USA). Antibodies used for Western blotting were: anti-HIF1AN (Sangon Biotech, Shanghai, China), anti-VEGF (Sangon Biotech, Shanghai, China), anti-HIF-1α (Sangon Biotech, Shanghai, China) and anti-Tubulin (Sangon Biotech, Shanghai, China). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (Santa Cruz, USA) were used. Finally, the membranes were treated with ECL reagents kit and exposed to X-ray film to detect the protein bands. Relative expression of relevant proteins were quantified and normalized to protein Tubulin.

Total protein was extracted by total protein extraction kit (KeyGEN BioTECH, Jiangsu, China), the concentration was assessed and the samples were boiled for 5 min. Then, proteins were separated by electrophoresis and transferred to the PVDF membrane, and blocked with a blocking solution (Sigma, USA) at room temperature for two hours. Next, samples were incubated with anti-HIF-1α (1:500, Sangon Biotech, Shanghai, China) and anti-ACTB (1:500, Sangon Biotech, Shanghai, China) antibody overnight at 4 °C, and then with anti-rabbit IgG, HRP-linked antibody (1:1000, CST, USA) at room temperature for 1 hour. Finally, samples were analyzed using a gel imager.