Journal: Journal of Cellular and Molecular Medicine
Article Title: Anti‐human CD 9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins
doi: 10.1111/jcmm.14334
Figure Lengend Snippet: Characterization of CD 9 Fab. A, Cell surface immunofluorescence on native FEMX ‐I cells. FEMX ‐I cells were surface labelled in the cold with CD 9 Fab at different concentrations as indicated (μg/mL), PFA ‐fixed and incubated with either anti‐Fab (top panels) or anti‐Fc (bottom panels) specific secondary conjugated to a fluorochrome (green). Nuclei were counterstained with 4′‐6‐diamidino‐2‐phenylindole ( DAPI ). B, Cell surface immunofluorescence on CD 9‐depleted FEMX ‐I cells. Native FEMX ‐I cells and CD 9 sh RNA ‐transduced cells were surface‐labelled in the cold with CD 9 Fab (top panels) or CD 9 Ab (bottom panels) at different concentrations (μg/mL), as indicated, PFA ‐fixed and incubated with anti‐Fab or anti‐Fc specific secondary conjugated to a fluorochrome (green) respectively, prior to DAPI staining. Note that under these conditions, about 15% of infected cells still express CD 9 in a proportion similar to native cells (asterisks). Scale bar, 25 μm. C, Immunoblotting. Detergent cell lysate (100‐μg protein) prepared from melanoma FEMX ‐I cells was probed using Fab CD 9 and horseradish peroxidase‐coupled anti‐Fab specific secondary antibody. β‐actin was used as control. Position of prestained molecular weight markers ( kD a) are indicated. Bracket, CD 9 immunoreactivity. D, Flow cytometry. FEMX ‐I cells were surface labelled with either CD 9 Fab (10 μg/mL, top) or CD 9 Ab (10 μg/mL, bottom) followed by fluorochrome‐conjugated anti‐Fab or anti‐Fc specific secondary antibody respectively. E, CD 9 Fab inhibits the cell binding of native CD 9 Ab. FEMX ‐I cells were sequentially labelled with CD 9 Fab at different concentrations as indicated (μg/mL) followed by CD 9 Ab (10 μg/mL) and fluorochrome‐conjugated anti‐Fc specific secondary antibody. Samples were analysed using flow cytometry. The median fluorescence intensity ( MFI ) is indicated. As negative and background controls, primary Ab (D) or CD 9 Ab (E) was omitted
Article Snippet: Proteins were separated using either 12% SDS‐PAGE gel (Figure and Figure ) or a precast gel (see above; Figure ) along with the Trident prestained protein molecular weight ladder (GeneTex, Irvine, CA) and transferred overnight at 4°C to a nitrocellulose membrane (Thermo Fisher Scientific) or poly(vinylidene difluoride) membrane (Millipore, Bedford, MA: pore size 0.45 μm).
Techniques: Immunofluorescence, Incubation, Staining, Infection, Western Blot, Control, Molecular Weight, Flow Cytometry, Binding Assay, Fluorescence
