Ab10972

Immunofluorescent staining for CD34, CD133, VEGFR2, and β-catenin was conducted using EPCs after two passages. Briefly, these cells were plated in 12-well plates (1× 10⁴/well) until 80% confluent, at which time they were fixed for 15 min using 4% paraformaldehyde (PFA), then permeabilized with 0.1% Triton X-100 for 20 min, and blocked for 30 min using 10% goat serum. These cells were then incubated overnight with mouse monoclonal anti-CD34 (12,034,042,1:50; eBioscience, USA), goat polyclonal anti-VEGFR2 (ab10972, 1:500; Abcam), rat monoclonal anti-CD133 (12,133,182, 1:100; eBioscience, USA), and/or mouse monoclonal anti-Beta-Catenin (138,400, 1:500; ThermoFisher) at 4 °C. Samples were then washed, probed for 1 h with secondary antibodies at 37 °C, and Nuclei were labeled with DAPI (C0065, Solarbio, China). Samples were then combined with 100 μl of anti-fluorescence attenuating mount, after which they were evaluated with an Intelligent Full-Automatic Fluorescence Microscopy Imaging System (Invitrogen™ EVOS™ FL Auto 2, Thermo Scientific, USA).

After cells treated with/without 100 ng/ml rhVEGF, 100 nM apatinib or 100 ng/ml rhVEGF + 100 nM apatinib, cells were lysed using lysis buffer (Cell Signaling Technology, Danvers, USA) to extract total protein. Protein lysates were separated by 10% SDS-PAGE, followed by transfer to nitrocellulose membranes. The membrane was then blocked with 5% milk diluted in PBS at room temperature for 1 h, followed by incubated with 1:1000 VEGFR2 antibody (ab10972, Abcam, Cambridge, MA, USA),1:5000 p-VEGFR2 (ab38473, Abcam), 1:2000 p-MEK (2338, CST), 1:1000 MEK (4694, CST), 1:2000 p-ERK1/2 (4370, CST), 1:1000 ERK (4695, CST), 1:2000 slug (ab51772, Abcam), 1:3000 Snail (ab53519, Abcam), 1:2500 MMP9 (ab38898, Abcam), 1:1500 P-AKT (ab81283, Abcam), 1:1500 AKT (ab179463, Abcam) and 1:5000 GAPDH antibody (ab8245, Abcam) overnight at 4 °C separately. Once primary antibodies were washed, membrane was incubated with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Sangon Biotech, Shanghai, China). Protein bands were detected by incubating the membrane with Western Bright enhanced chemiluminescence working solution (Advansta, Menlo Park, CA, USA). The film (Kodak XBT-1, Carestream, Xiamen, China) was scanned with Bio-rad Gel Doc XR+ (BIO-RAD, Shanghai, China).

Tissue protein of heart and spleen was lysed by the RIPA lysate (50:1) and tissue disrupter and quantified by the BCA method. The quantified homogenate was added to loading buffer, and the mixture was boiled and denatured at 99°C. Each lane was loaded with 10 ul of proteins. After electrophoresis at 100 V for 1–1.5 h, proteins were transferred to PVDF membranes at 300 mA for 1–1.5 h. Afterwards, the membrane blocked with 5% skim milk for 1–1.5 h at room temperature, incubated on a shaker, and washed with TBS-T. Western blot analysis was conducted using anti-AT1 (ab18801; Abcam, United States), anti-MCP-1 (ab25124, Abcam, United States), anti-CCR2 (PAI-27409), anti-TGF-β1 (3711s, Cell Signaling Technology, Germany), anti-Smad3 (ab28379, Abcam, United States), anti-MMP2 (ab86607, Abcam, United States), anti-Col III (ab7778, Abcam, United States), anti-VEGF (ab10972, Abcam, United States), anti-CD31 (ab24590, Abcam, United States) and anti-GAPDH (ab8245, Abcam, United States) at 4°C overnight. After incubation with the appropriate secondary antibodies at 37°C for 2 h at room temperature, the membrane treated with ECL for 1 min at room temperature. The final expression of each protein was normalized by GAPDH and grayscale analysis was performed using Image J software.