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Fig. 5. Localization of uPAR and PN-1 to sites of cell adhesion in monocytic cells. (A) U937 cells were differentiated into monocytic lineage and preincubated with a combination of uPA (50 nM) and rhodamine labeled PN-1 (3 µg/ml). After extensive washing the cells were allowed to adhere to VN- or FN-coated slides. Adherent cells were fixed and immunostained with anti-uPAR (rabbit polyclonal) antibody followed by FITC-coupled anti-rabbit IgG. Confocal microscopy was used to determine the location of the antigens in serial sections of the cells. The top, middle and bottom sections from adherent cells on VN and FN are shown. Left column shows uPAR labeling, the middle column, PN-1 and the right column shows the overlay of the two. Similar results were obtained in three separate experiments. (B) The fluorescence intensity of uPAR and PN-1 on VN- or FN-coated slides was quantified in 0.2 µm serial sections. Relative fluorescence with anti-uPAR IgG on a VN (open triangle) or FN substratum (solid triangle) and PN-1-Rhodamine on a VN (open square) or FN substratum (solid square) is shown.
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