PDI was localized intracellularly and not at the cell surface. (A) Cellular localization of PDI by immunofluorescence confocal microscopy. Nonpermeabilized and permeabilized MDA-MB-231 cells were immunostained with rabbit anti-human PDI or monoclonal anti-human PDI, monoclonal anti-human TF (10H10), or rabbit anti-calreticulin antibody, followed by Oregon Green–labeled anti-rabbit and Rhodamine red-conjugated anti-mouse antibodies as secondary reporter antibodies. (B) Nonpermeabilized and permeabilized MDA-MB-231 cells were stained with anti-PDI IgG or anti-TF immunoglobulin G (IgG), followed by FITC-conjugated secondary antibodies. In the right panel, MDA 231 cells were treated with HgCl2 (25 μM), PAR-1 or PAR-2 agonist peptides (50 μM), thrombin, or FVIIa (10 nM) for 30 minutes before the cells were stained with PDI antibodies. The cells were analyzed by flow cytometry. (C) MDA-MB-231 cells were labeled with cell-impermeant NHS-SS-biotin or a control vehicle. Total cell extracts and cell extracts subjected to immunoprecipitation with streptavidin-agarose were immunoblotted with anti-biotin, anti-PDI, or anti-TF antibodies. TC indicates total cell extract; IP, immunoprecipitated; and kDa, kilodaltons.
