Dyc5079 2

HUVECs were seeded in 12-well plates with EBM-2 Basal medium (CC-3156, Lonza) supplemented with EGM-2 SingleQuots Supplements (CC-4176, Lonza) for 24 h. Cells were then stimulated with 5 ng/ml bFGF (Biolegend) or 5 ng/ml bFGF in combination with either 50 or 100 µm K5 for 60 min. The cells were rinsed with PBS and lysed in cell lysis buffer containing IC diluent #12 (Reagent diluent concentrate 2 DY995, R&D Systems, Minneapolis, MN, USA, in distilled water), plus 10 µg/ml aprotinin (Ref.4139, R&D Systems, Minneapolis, MN, USA) and 10 µg/ml leupeptin (Ref.1167, R&D Systems, Minneapolis, MN, USA). FGFR1 phosphorylation was measured by a sandwich ELISA (DYC5079-2, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Differentiated THP-1 cells were incubated in HH enriched media (0.1 mg/mL of Hb:Hp (H0267 and SRP6507, Sigma Aldrich) in complete medium) over 6 days. At day 7, 10 µg/mL and 100 µg/mL of PC-mAb was added to the medium and incubated overnight.
Cells were scraped and homogenized in modified RIPA buffer containing sodium-orthovanadate and protease inhibitors. Proteins were separated by SDS-PAGE (4–15%) and transferred to nitrocellulose. Blots were incubated with antibodies against CD163 (93498, Cell Signaling). A peroxidase conjugated secondary antibody was used (31462, 31400, ThermoFisher Scientific). Proteins of interest were imaged with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34580, ThermoFisher Scientific) and the ChemiDoc™ Touch Imaging using System (1708370, Bio-Rad Laboratories). β-actin (ab8220, Abcam) was used as internal control and blots were quantified with Image J.
Cell supernatant was also collected and VEGFA was measured by a sandwich ELISA (DYC5079-2, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.