Figure 1.

ProNCAD is expressed on the surface of invasive tumor cells. (A) Western blot analysis of NCAD levels in U343 and U251 glioma cells and in WM115 and WM266 melanoma cells using an NCAD cytoplasmic antibody. Anti-actin immunoblot is shown as an independent loading control. Results were quantified by densitometric analysis. (B) Left panel: Western blot analysis of cell surface biotinylated proteins from U343 and U251 cells. A cell surface biotinylation assay was carried out to determine the proportion of proNCAD on the surface of U343 and U251 cells. Experiments were carried out with 90 or 60 µg of total protein, and NCAD cytoplasmic and proN antibodies were used to detect total NCAD and specifically proNCAD, respectively. Anti-Na⁺/K⁺-ATPase blot is shown as an independent loading control for both total cell lysates and biotinylated fractions. Anti-ERK blot is shown as a control for surface biotinylation. The proportion of surface to total proNCAD was determined by densitometry. Values are means ± SEM. *P < .01. Right panel: Western blot analysis of cell lysates of WM115 and WM266 melanoma cells. NCAD cytoplasmic antibody detected levels of total NCAD protein in the melanoma cells. The proN antibody specifically detected substantially higher levels of proNCAD in WM266 cells. L cells overexpressing NCAD (LN cells) served as a positive control for properly processed NCAD. Anti-actin blot is shown as a loading control. (C) Immunocytochemistry demonstrating intracellular localization of proNCAD in permeabilized tumor cells (top panels) and substantial surface localization in U251 and WM266 nonpermeabilized (bottom panels), live tumor cells. Images were taken using the Zeiss LSM 510 confocal microscope with the Zen image acquisition software and a 60x oil immersion objective. All cells were visualized in the same plane of focus between comparisons. Bar, 10 µm. (D) Cell aggregation assay of glioma and melanoma cells in the presence of calcium during a 40-minute time course. The number of single cells was counted during cell aggregation under a light microscope, and the percentage of single cells during the aggregation assay was determined by the index N_t/N₀, where N_t is the total number of single cells after a certain incubation time and N₀ is the total number of single cells at the initiation of incubation. Values are means ± SEM. Results in each panel are representative of three independent experiments.