FVIIa-mediated signaling impairs TNF-α-induced TRAF2 recruitment to TNFR1 and not colocalization of TNFR1 with caveolin-1. HUVECs were treated with a control vehicle or FVIIa (100 nM) for 1 hour and then stimulated with TNF-α (5 ng/mL) for 5 minutes in serum-free medium. (A-B) Cell lysates were immunoprecipitated with TNFR1 antibodies, and the immunoprecipitates were probed for the presence of caveolin-1 (A) or TRAF2 (B) using western blotting with specific antibodies. The bottom panels show quantitated data from densitometric analysis of immunoblots from 3 to 4 independent experiments (ns, not significant, *P < .05). (C). HUVECs cultured on a glass coverslip were treated as described above and subjected immunofluorescence confocal microscopy to probe for localization of TNFR1 (green) or TRAF2 (red). Panel Ci shows representative immunofluorescence micrographs, and panel Cii shows colocalization coefficient calculated from the analysis of ≥30 cells (***P < .001). Please note that because of differences in the intensity of red and green fluorescences, colocalization does not necessarily give a yellow color signal in merged images and therefore it can be difficult to discern differences visually among the images. Scale bar indicates 100 µm.