Ecl plus reagent

Total protein of the tissues and transfected cells was extracted using a protein extraction kit (Beyotime Institute of Biotechnology), and protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg) were separated via SDS-PAGE on 10% gel, and subsequently transferred to a polyvinylidene difluoride membrane. After blocking for 2 h in 5% skimmed milk at room temperature, the membranes were incubated with the following primary antibodies overnight at 4°C: Anti-HOXB2 (cat. no. ab220390; Abcam), anti-phosphorylated (p)-PI3K (cat. no. ab32089; Abcam) anti-p-AKT (cat. no. 4058; Cell Signaling Technology, Inc.), anti-caspase-3 (cat. no. 9662; Cell Signaling Technology, Inc.), anti-cleaved-caspase-3 (cat. no. 9664; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.). The primary antibodies were used at 1:1,000 dilution. After washing three times with TBS with 0.3% Tween-20, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) at 37°C for 30 min. Protein expression was visualized using ECL-Plus reagent (Santa Cruz Biotechnology, Inc.) and analyzed with the ChemiDoc™ XRS imaging system (Bio-Rad Laboratories, Inc.). GAPDH was used as the loading control.

BMECs were lysed with cold radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA) for 30 min and protein concentrations were analyzed by the BCA method. Whole-cell lysates (30 µg) were fractionated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked with 5% bovine serum albumin (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 30 min and incubated overnight at 4˚C with primary antibodies against VEGFA (1:1,000; anti-rabbit), HIF-1α (1:1,000; anti-rabbit), IL6 (1:1,000; anti-mouse), β-actin (1:1,000; anti-rabbit). The membranes were subsequently incubated with a secondary antibody (1:10,000; goat anti-rabbit or goat anti-mouse antibody) at 37˚C for 1 h. The blots were visualized using an ECL-Plus reagent (Santa Cruz Biotechnology, Inc.) and analyzed using Quantity One System image analysis software version 4.6.2 (Bio-Rad Laboratories, Inc.).