Table 2.
Cryoimmunogold quantitation of ligand-induced redistribution of uPAR
| Ligand | Plasma-membrane (%) | Vesicles and vacuoles (%)a | Endosomes (%)b | Lysosomes (%)c | Total gold particle count | No. of cell profiles counted |
|---|---|---|---|---|---|---|
| DFP-uPA | 78.0 ± 2.8 | 12.5 ± 0.7 | 9.0 ± 1.4 | 2376 (n = 3) | 21 | |
| 78.0 ± 2.0 | 16.3 ± 2.1 | 6.0 ± 0.0 | 2478 (n = 2) | 17 | ||
| uPA/PAI | 60.0 ± 15.1 | 28.8 ± 13.2 | 14.0 ± 4.0 | 3523 (n = 3) | 17 | |
| 56.3 ± 16.4 | 32.0 ± 13.2 | 11.3 ± 2.9 | 1580 (n = 3) | 20 |
Nonpolarized MDCK cells that had internalized prebound DFP-uPA or uPA:PAI-1 for 150 min were processed for cryoimmunogold double labeling using the antibody combinations uPAR/MPR or uPAR/LAMP-1, respectively. Antibodies were detected with 10 or 15 nm gold-conjugated protein A and quantitated. Means and SDs from two or three independent experiments are shown (multiple grids from each experiment were counted).
Percentage of total uPAR gold particle count located in vesicles and vacuoles containing labeling exclusively for uPAR.
Percentage of total number of uPAR gold particles colocalized with at least two MPR gold particles.
Percentage of total number of uPAR gold particles colocalized with at least two LAMP-1 gold particles.