Binding of FX to cellular SR-AI. (A-D) THP1-derived macrophages were preincubated with PBS (A), nonspecific IgG (B), or a polyclonal anti–SR-AI antibody (C) and further incubated with 10 µg/mL Alexa 488–labeled FX (1 hour at 37°C). Images were acquired in widefield microscopy and quantified for FX fluorescence (D). (E-I) Immunofluorescent staining of SR-AI (red) and FX (green) was performed in nontransfected HEK-293 cells (E and G, respectively) or HEK-293 cells transfected with pcDNA6/hSR-AI (F and H, respectively) incubated with 10 µg/mL FX (1 hour at 37°C). Images were acquired in widefield microscopy and subsequently quantified for FX fluorescence (I). Data are presented in mean pixel intensity per cell (D,I). Boxes represent the median and 25th to 75th percentile, and bars represent the 10th to 90th percentile (at least 5 different fields per experiment in 3 different experiments). (J) Representative images of double immunostaining for FX and SR-AI in HEK-293 pcDNA6/hSR-AI analyzed using confocal microscopy. Nuclei and polymerized actin were counterstained using DAPI (blue) and Alexa 647–labeled phalloidin (magenta), respectively. Objective, ×63; bars represent 10 µm; Z depth is 0.4 µm (J) and arrows indicate area of colocalization. ***P < .001, respectively, in the Mann-Whitney nonparametric unpaired statistical test.