Anti hif 1 antibody

ChIP assays were performed using the EZ-ChIP ™ Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The chromatin complexes were immuno-precipitated with anti-HIF-1 antibody (Abcam). Then the ChIP Assay Kit (Beyotime) was used to purify DNA samples, and the purified DNA was analyzed by qPCR. The primer pairs used to amplify the HIF-1 binding site in the NPTX2 promoter was: f: 5′- GTGACACTGCGGGCCCT-3′ and r: 5′- CGTCCGGCGCCTGGTCCT-3′.

ChIP assays were performed using the EZ-ChIP™ Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The chromatin complexes were immunoprecipitated with anti-HIF1 antibody (Abcam), and the purified DNA samples were analyzed with qPCR using primer pairs for the HIF-1-binding site in the PLOD1 promoter f: 5′- ATCCGTGCCCACCCTCA-3′ and r: 5′- AGAAAGAGGGCGGTGAGA-3′. All reactions were performed in triplicate.

Following treatment or transfection, culture supernatants were removed, cells or tissues were lysed using radio immunoprecipitation assay buffer (RIPA, Beyotime, China), and protein was harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extracts were separated in 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, USA). After incubation with a high affinity anti-c-Myc antibody (1:1000, Abcam), anti-ELK1 antibody (1:1000, Abcam), anti-cdc25c antibody (1:1000, Abcam), anti-hif1 antibody (1:1000, Abcam), anti-c-Jun antibody (1:1000, Abcam), anti-Mdm2 antibody (1:1000, Abcam), anti-JunB antibody (1:1000, Abcam), anti-mct4 antibody (1:1000, Abcam), anti-pdk1 antibody (1:1000, Abcam), anti-pdk4 antibody (1:1000, Abcam), anti-GLUT1 antibody (1:1000, Abcam), anti-GLUT4 antibody (1:1000, Abcam), anti-Ubiquitin antibody (1:1000, Abcam) or anti-β-actin antibody (1:2000, Cell Signaling Technology, USA) in Primary Antibody Dilution (MB9881, Dalian Meilun Biotechnology Co., Ltd), the membranes were incubated with a secondary antibody (1:5000, Cell Signaling Technology, USA). After washes, signals were detected using FDbio-Femto ECL (Fudebio, Hangzhou, China) and a chemiluminescence system (Bio-Rad, USA). All images were analyzed using Image Lab software.