Human igg1 fc fragment

HUVECs were seeded in six-well plates, grown until 80% of confluence, starved for 2 h, pretreated for 20 min with UniPR1331 or 0.3% DMSO, stimulated with 30 ng/ml VEGF for 30 min, washed with PBS, harvested, and centrifuged. Pellets were suspended in flow cytometry staining buffer and blocked with human IgG1-Fc fragment for 20 min (Millipore). Human VEGFR2-PE-conjugated antibody or mouse IgG-PE-conjugated isotype control antibody (R&D systems) was added and incubated at 25 °C. Unbound antibody was removed by washing and VEGFR2 internalization was revealed by flow cytometry analysis (Guava easyCyte 5, Millipore). Data were analyzed with FlowJo software (Ashland, OR).

Recombinant human CD137-Fc fusion protein was purchased from R&D Systems (Minneapolis, MN, USA) while human IgG1 Fc fragment was purchased from Millipore (Billerica, MA, USA). GM-CSF, M-CSF and IL-4 were purchased from Peprotech (Rocky Hill, NJ, USA). IFN-γ was obtained from R&D Systems. Lipopolysaccharide (LPS) derived from E. coli of serotype 0111:B4 was obtained from Sigma-Aldrich (St. Louis, MO, USA). Resiquimod (R848) was purchased from Invivogen (San Diego, CA, USA).