Figure 6.

FHR-1 binds to apoptotic and necrotic cells. (A) Binding of 300 nM (black lines) recombinant FHR-1 to live and necrotic retinal pigment epithelial cells (ARPE-19), human umbilical vein endothelial cells (HUVECs), and Jurkat T cells in DPBS with Ca²⁺/Mg²⁺ was measured by flow cytometry using polyclonal anti-FH Ab and a corresponding secondary Ab. Gray histograms indicate antibody binding controls without FHR-1. (B) Binding of native FHR-1 from NHS to necrotic cells. Necrotic cells were incubated with NHS, washed, and then lysed. Cell lysates were analyzed by SDS-PAGE and western blot using polyclonal anti-FH Ab. Representative blots of three experiments are shown. (C) FHR-1 binding to apoptotic cells was measured on staurosporine-treated Jurkat T cells. The dot plot (left panel) shows the ratio of early and late apoptotic Jurkat cells after 1-day staurosporine treatment and labeling with Annexin V-PE and 7-AAD. After gating, histograms show 300 nM FHR-1 binding to early (middle panel) and late (right panel) apoptotic Jurkat T cells. Representative data of three experiments are shown.