Cellular distribution of EPCR. (A) Confluent monolayers of HUVECs, nonpermeabilized or permeabilized with 0.1% Triton X-100 for 10 minutes, were immunostained with EPCR mAbs (JRK1500, 10 μg/mL), followed by Rhodamine Red-conjugated anti–mouse IgG. Immunofluorescence was analyzed by confocal microscopy. (Left) Images from a single plane of z-stack. (Right) Three-dimensional reconstructed composite images of all z-stacks. (B) CHO-EPCR cells were immunostained with EPCR mAbs as described in panel A. (C) HUVECs (top panel) and CHO-EPCR cells (bottom panel) were first treated with either control vehicle or mβCD (10 mM) for 30 minutes (to deplete the membrane cholesterol) and then permeabilized with 0.1% Triton X-100 for 10 minutes. The permeabilized cells were immunostained with polyclonal anti–human caveolin-1 and EPCR mAbs followed by Oregon Green–labeled anti–rabbit IgG and Rhodamine Red-labeled anti–mouse IgG as secondary reporter antibodies. (Right, merge) Overlay of caveolin-1 and EPCR staining. The images shown were composite images. (D) CHO cells were transfected transiently with full-length EPCR-GFP or EPCR lacking N-terminal signal peptide (EPCR Δsp)–GFP fusion construct, and the expression of green fluorescent fusion product was analyzed by confocal microscopy. (E) CHO-EPCR cells were permeabilized and immunostained with polyclonal anti–γ-tubulin and EPCR mAbs. (Insets) The magnified view of the boxed regions. Arrow represents separation of green and red fluorescence, which indicates that γ-tubulin is not colocalized with EPCR. The presence of EPCR around γ-tubulin indicates that EPCR is localized in the pericentriolar region. (F) CHO-EPCR cells were immunostained either with polyclonal anti-rab11 and EPCR mAb (top panel) or transiently transfected with rab11-GFP and then immunostained with EPCR mAb (bottom). (Insets) Magnified view of the boxed regions. It is known that rab11 also associates with other endosomal compartments and secretory vesicles19,–21 ; therefore, rab11 antibodies, in addition to the REC, also stained other intracellular compartments. (G) CHO-EPCR cells (top panel) or HUVECs (bottom panel) were treated with AF555-transferrin (300 nM) for 1 hour and then immunostained with EPCR mAbs. Arrows in the right panel represent the colocalization of EPCR and AF555-trasferrin in the REC. Scale bar represents 15 μm. The REC is not visual in all cells as its position could vary from cell to cell. The REC that is positioned on top of the nucleus may give a false impression that it is localized in the nucleus.
