Characterization of Axl MAb173. (A-C) Effect of MAb173 treatment on the invasion of HUVEC (A), LTC (B), and KS-IMM (C) was analyzed by transwell invasion assay. The experiment was performed in triplicate. An unrelated isotype control antibody (Ctrl IgG) was used as a negative control. The cells that accomplished invasion were counted in 10 individual high-powered fields for each membrane under a light microscope, and the numbers were shown below each representative picture. (D) LTC was treated with Ctrl IgG and MAb173 for the indicated time. The whole-cell lysates were subjected to Western blot with antibodies against Axl and pAKT. The relative expression level was quantitated by ImageJ, normalized to β-actin and is shown below each panel. (E) LTC cells were incubated with 10 μg/mL biotinylated MAb173 at 37°C for 15 minutes or 1 hour, or 4°C for 1 hour. The cells were fixed and MAb173 was localized with streptavidin-fluorescein isothiocyanate (green). Nuclei were counterstained with DAPI (blue). Images were taken with a Carl Zeiss confocal microscope (100× objective) and LSM software. (F) The scheme of Axl truncation variants (Fc fusion for all) is shown on the top left. The C-terminal amino acid of Axl in each construct was indicated. The expression of these constructs was analyzed by Western blot using antibody against Fc and was shown on the top right. The epitope of MAb173 was determined by ELISA (bottom panel), as described in “ELISA.” The experiment was performed in duplicate.