Plates were coated with DPBS or FHR-1 (5 μg mL−1), and 1 × 105 monocytes were added in CM containing 10% NHS with or without addition of the PLC inhibitor U73122 (10 μM, Abcam) or the NF-κB inhibitor BAY11-785 (30 μM, Sigma-Aldrich). After incubation for 20 h at 37 °C in an atmosphere containing 5% CO2, the cells were washed twice with DPBS, followed by the addition of Factor VIIa substrate (0,3 mg mL−1, Sigma-Aldrich) in DPS containing 20% NHS and Mg2+/Ca2+ (Biowest). After 5 min, absorbance was measured at 405 nm. For EMR2 blocking, an anti-EMR2 antibody (10 μg mL−1, AF4894, R&D Systems) was added, and the cells were incubated for 5 h at 37 °C. The cells were washed twice, followed by the addition of Factor VIIa substrate (0.2 mg mL−1, Sigma-Aldrich) in DPBS containing 10% NHS and Mg2+/Ca2+ (Biowest). After 85 min, absorbance was measured at 405 nm.
Irmscher S., Zipfel S.L., Halder L.D., Ivanov L., Gonzalez-Delgado A., Waldeyer C., Seiffert M., Brunner F.J., von der Heide M., Löschmann I., Wulf S., Czamara D., Papac-Milicevic N., Strauß O., Lorkowski S., Reichenspurner H., Holers M.V., Banda N.K., Zeller T., Binder E.B., Binder C.J., Wiech T., Zipfel P.F, & Skerka C. (2021). Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease. Scientific Reports, 11, 22511.
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