Targeted cytotoxicity of the 90G4 immunotoxin in migratory cells.

<p>Live imaging of 90G4 immunotoxin administrated either (A–C) concomitantly or (D–F) one day after SVEC4-10 cell seeding. For each condition, samples were seeded in triplicate. (B, E) Phase contrast images were taken every 3 h and analyzed to calculate confluency (%). Values and error bars correspond to mean and standard error, respectively. (C, F) Images were taken at the indicated time during live imaging.</p

90G4 monoclonal antibody recognizes the CD321 antigen.

<p>(A) Expression profile in endothelial cell lines. Indicated endothelial cell lines were tested by staining with control (gray) or 90G4 antibodies (black). Data of FITC fluorescent intensity (FL1) indicated in histogram. Data presents three independent experiments. (B) Biochemical profiling of the molecular weight of the putative antigen. SVEC4-10 cells were surface-biotinylated with Sulfo-NHS-Biotin and lysed in NP40 buffer, followed by immunoprecipitation with 90G4 or IgG2a, к control antibodies. After SDS-polyacrylamide gel electrophoresis (PAGE), the putative antigen was visualized by probing the streptavidin-HRP (Str-HRP) conjugate by chemiluminescence. The molecular weight of the detected protein was 35 kDa. (C) Identification of 90G4 antigen. Amino acid sequence of CD321 proteins were indicated with the two of underlined peptide sequences that are detected by LC-MS/MS analysis. (D) Specific immunoreactivity of 90G4 antibody against CD321. The expression vectors encoding CD321, CD322, or CD323 cDNA were transfected into CHO cells, which were then subjected to flow cytometry analysis. Data are presented in contour plot (top) or overlaid in histogram (bottom) of FITC signal intensity of sample (black) or control (gray).</p