Ab233135

Western blot assay was used to detect protein expression levels. Proteins were extracted using RIPA total protein lysate (cat. no. P0013C, Beyotime, Inc, China) following the manufacturer's instructions. Proteins from each sample were separated by electrophoresis on 10% SDS-PAGE gels and transferred onto PVDF membranes (cat. no. FFP39, Beyotime, Inc, China). After incubation with blocking solution (cat. no. P0252, Beyotime, Inc, China), the PVDF membranes were separately incubated with anti-BCL-2, anti-BAX, anti-cleaved caspase-3, anti-GAPDH, anti-HIF-1α, anti-HIF-2α, PGK-1, VEGF, EPO, caspase-3 and p53 (cat.no. ab32124, ab32503, ab32042, ab9485, ab51608, ab243861, ab233135, ab32152, ab226956, ab32351, ab32389, Abcam, Inc, USA,1:1000) at 4°C overnight. After the blots were washed, they were incubated with HRP-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (cat.no. ab6721, Abcam, Inc, USA,1:2000) for 1.5 h and detected by ECL like Western reagent (cat. no. P0018S, Beyotime, Inc, China) with a chemiluminescent imaging system (ChemiDoc MP, Bio-Rad, Inc, USA). For the consideration of saving primary antibodies, the PVDF membranes were cut prior to hybridisation with primary antibodies, thus the images of full length membranes were absence in this manuscript.