Anti vegfr2 antibody

Western blot analysis was carried out for VEGFR2 using an anti-VEGFR2 antibody (Abcam Co.) as the primary antibody. The cells were collected and lysed by RIPA lysis buffer (Sigma-Aldrich Corp., St. Louis, MO, USA). Total protein was extracted and stored at -80˚C. The extracts were then mixed with 6× sodium dodecyl sulfate (SDS) buffer and boiled for 4 minutes. Samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% (w/v) skim milk in PBS that contained 0.1% Tween-20 for 1 hour at room temperature, washed with PBS, and probed with primary antibodies overnight at 4˚C. Membranes were washed again with PBS and incubated at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (Abcam Co., UK) for 1 hour. Proteins were visualized with an enhanced chemiluminescence detection kit (Amersham Bioscience, Buckinghamshire, England). Actin (a goat polyclonal antibody) was used as the internal control.

For primary CD4⁺ T-cell isolation, PBMCs were acquired utilizing Ficoll Paque Plus (GE Healthcare, USA) gradient cell separation according to the manufacturer’s instructions. Next, the CD4⁺ T Cell Isolation Kit, human (Miltenyi Biotec, Germany) was utilized to isolate CD4⁺ T cells. Then, the cells were resuspended at 1 × 10⁶ cells/mL and cultured in advanced RPMI1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% 2-mercaptoethanol (2-ME; Gibco) and 1% penicillin–streptomycin (Gibco) in 48-well plates precoated with 2 μg/mL anti-human CD3 antibody (BioLegend, USA). For VEGF-R-related assays, after incubation with 5 μg/mL anti-VEGF-R1 antibody (R&D Systems, USA) and/or 10 μg/mL anti-VEGF-R2 antibody (Abcam, USA) for 1 h, CD4⁺ T cells were treated with blank (CON) or 15 ng/mL VEGF-A (R&D Systems, USA) for 2 days respectively. GolgiPlug Protein Transport Inhibitor (BD Bioscience, USA) was administered for the last 6 h to detect the cytotoxic molecules. Regarding AKT/mTOR inhibition assays, CD4⁺ T cells were treated with blank (CON), 15 ng/mL VEGF-A, VEGF-A + 10 μM MK-2206 2HCl (AKT inhibitor, MedChemExpress, USA), VEGF-A + 100 nM rapamycin (mTOR inhibitor, Selleck, China) or VEGF-A + 10 μM MK-2206 2HCl + 10 μM MHY1485 (mTOR activator, MedChemExpress, USA) for 2 days in the presence of GolgiPlug Protein Transport Inhibitor for the last 6 h.