Primary antibodies against vegf

Total protein was extracted from peritoneal tissues of rats. The concentration of protein was determined using the BCA Kit (Boster Bio-Engineering Co. Ltd., Wuhan, China). The protein samples were boiled with buffer solution at 95°C for 10 min, at a concentration of 40 µg/per well. The protein samples were separated by 10% SDS/PAGE (Boster Bio-Engineering Co. Ltd., Wuhan, China) with a voltage from spacer gel at 80 V to separation gel at 120 V and were then wet-transferred on to PVDF membranes with a constant voltage of 100 mV for 90–120 min. The membranes were sealed with 5% BSA at room temperature for 1 h, and then incubated with primary antibodies against VEGF (1:1000; Abcam Inc., Cambridge, MA, U.S.A.) and β-actin (1:1000; Abcam Inc., Cambridge, MA, U.S.A.) at 4°C overnight. After washing with TBS and Tween 20 (TBST) three times (5 min per wash), the membranes were incubated with the secondary antibody at room temperature for 1 h. Next, the membranes were washed three times with TBST (5 min per wash). The protein bands were visualized by chemiluminescence reagent. β-actin served as an internal reference indicator. The gray values of the protein bands were analyzed using ImageJ software.

Total RNA was isolated using RNA extraction kit (Takara Bio, China) and qPCR was performed. The following primers were used: VEGF A (sense); 5′- CAA​AGC​CAG​CAC​ATA​GGA​GAG​A-3′, and VEGF B (antisense); 5′-CTA​TCT​TTC​TTT​GGT​CTG​CAT​TCA​C-3′; rat actin A (sense); 5′-CCC​ATC​TAT​GAG​GGT​TAC​GC-3′, and rat actin B (antisense); 5′-TTT​AAT​GTC​ACG​CAC​GAT TTC-3’ (Takara Bio, China). EPC protein was extracted using cell lysis buffer (Beyotime, China) and VEGF protein expression was determined using western blotting. Total cell protein was extracted using RIPA buffer containing phosphatase and protease inhibitors (PMSF) (Beyotime, China). Protein concentration in the supernatant was measured using a BCA protein kit (Boster Bio, China). Protein was separated using SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) (Millipore, United States) membrane. The membrane was incubated overnight at 4°C with primary antibodies against VEGF (1:2,000) and β-actin (1:1,000) (Abcam, United States). The PVDF membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000) for 30 min. The membranes were washed again, treated with chemiluminescence (ECL) solution (Millipore, United States), and visualized by exposure to film. The expression of each protein relative to β-actin was determined using ImageJ software.