Geldoc it310

Electrophoresis The obtained PCR product was separated by electrophoresis (Biometra Horizon 20.25 with Consort EV243 power pack) on a 1% agarose gel with 4 μl ethidium bromide (10 mg/ml stock solution, Sigma-Aldrich) for 35 minutes (160 V). The products were visualised by a gel documentation system with UV transilluminator (UVP GelDoc IT310).

Tissues were homogenized with liquid nitrogen. Cells and tissues were then lysed by lysis buffer for 10 min on ice and then, centrifuged at 12,000 rpm for 15 min at 4°C. Supernatants were mixed with loading buffer and underwent SDS-PAGE to separate proteins. Proteins were then transferred into the PVDF membrane. The membranes were blocked by 5% nonfat milk in Tris-buffered saline (TBS) and then, incubated with anti-β-Actin (1 : 1000, ab6276, Abcam, USA), anti-HIF1α (1 : 500, ab179483, Abcam, USA), anti-IDH2 antibodies (1 : 500, ab131263, Abcam, USA), and anti-Hydroxy-HIF1α (1 : 500, 3434, Cell Signaling Technology, USA) at 4°C overnight. After being washed for 3 times by 0.5% TBST, membranes were incubated with second antibodies at a dilution of 1 : 4000 at room temperature for 2 hours then washed by 0.5% TBST for 3 times. Blots were then quantified by electrochemiluminescence and visualized by Gel Imaging System (GelDoc-It310, UVP, USA).