Enhanced chemiluminescence plus kit

RIPA lysis buffer and protease inhibitor (Beijing Solarbio Science & Technology Co., Ltd.) were used at a ratio of 100:1 to lyse the tissues and cells to collect the supernatant. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Soluble proteins (40 µg) were separated using 8 and 12% SDS-PAGE gel and transferred onto PVDF membranes. Membranes were then blocked with 5% non-fat milk in TBS-0.1% Tween-20 (TBST) for 2 h at room temperature and incubated with a rabbit anti-human HIF-1α (1:1,000; cat. no. ab51608; Abcam), rabbit monoclonal anti-FOXO3a (1:1,000; cat. no. 12829S; Cell Signaling Technology, Inc.) and rabbit polyclonal anti-β-actin (1:2,000; cat. no. ab8227; Abcam) overnight at 4°C. The membranes were incubated at room temperature for 1 h and washed three times for 10 min with TBST. Subsequently, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000; cat. no. IH-0012; Beijing Dingguo Changsheng Biotechnology Co., Ltd.) for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence Plus kit (Beyotime Institute of Biotechnology) and an AI600 imaging system (GE Healthcare Life Sciences), and the relative band density was calculated using Adobe Photoshop 13.0 software (Adobe Systems, Inc.) (20 (link)).

cDNA was prepared by using oligonucleotide (dT), random primers, and a SuperScript First-Strand Synthesis System. PCR analysis was performed according to the manufacturer. β-actin was used as an internal control.
Western Blotting Proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membranes. And then blocked in 10% dry milk-TBST (20mM Tris-HCl [PH 7.6], 127mM NaCl, 0.1% Tween 20) for 1 h at 37°C. Following three washes in Tris-HCl pH 7.5 with 0.1% Tween 20, the blots were incubated with antibody (appropriate dilution) overnight at 4°C. Signals were visualized by enhanced chemiluminescence plus kit (Beyotime Biotechnology).