Simultaneous binding of rhAPC to neutrophil integrins and EPCR. (A) EPCR RT-PCR on human neutrophils. Reverse-transcribed cDNAs from human heart and lung served as positive controls (top). FACS analysis of cell-surface EPCR using mAb RCR252 (bottom). IgG control and fMLP-treated cells were exposed to fMLP for 30 minutes before staining with Rat IgG₁ isotype and RCR252, respectively. As a negative control (Untreated), cells were incubated without fMLP and labeled with RCR252. (B) Binding of 10 nM fMLP-treated neutrophils to immobilized FN in the presence of rhAPC (10 μg/mL) ± EPCR mAb (50 or 100 μg/mL). (C) A hypothetical model for rhAPC binding. Cells expressing hEPCR-mCFP and β₁-mYFP will exhibit FRET only when these 2 molecules are brought into close proximity (100 Å) after rhAPC binding. (D) Whole-cell lysates of HEK293 cells transiently transfected with hEPCR-mCFP and β₁-mYFP K562 were subjected to SDS-PAGE and Western blotting with the indicated antibodies. (E) Fluorescence images of transiently transfected HEK293 cells with hEPCR-mCFP and β₁-mYFP demonstrate membrane localization of CFP and YFP signals. (F) HEK293 cells were transfected with hEPCR-mCFP and β₁-mYFP in a delta T dish. FRET images under TIRF microscopy were extracted from videos (time above images, Videos S3Video 4. Time-lapse movie shows FRET image (highest red to lowest blue) of a hEPCR-mCFP and β1-mYFP transfected HEK293 cell (MOV, 429 KB)–S5). FRET was measured by sensitized emission method and analyzed by AutoQuant software. FRET signals are shown as rainbow colors (red indicates highest and blue, lowest).