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Blood, Vol. 104, Issue 4, 1183-1190, August 15, 2004
CD63 tetraspanin slows down cell migration and translocates to the endosomal-lysosomal-MIICs route after extracellular stimuli in human immature dendritic cells
Blood Mantegazza et al.
104: 1183
Supplemental materials for: Mantegazza et al, Vol 104, Issue 4, 1183-1190
Files in this Data Supplement:
- Figure S1. Controls for Figure 2 (JPEG file, 228 KB)
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Top left: DCs were sequentially incubated with FC-5.01, cy5-GAM (green), and cy3-GAR (red). Only cy5-GAM stained FC-5.01. Top right: DCs were sequentially incubated with FC-5.01, cy3-RAM (red), and cy5-RAG (green). Only cy3-RAM stained FC-5.01. Center left: DCs were incubated with isotype-matched control IgG2a followed by cy5-GAM. Center right: DCs were incubated with isotype-matched control IgG2a followed by cy3-RAM. Bottom left: DCs were incubated with control immunoglobulins followed by cy3-GAR. Bottom center: DCs were incubated with control immunoglobulins followed by cy5-RAG. Bottom right: DCs were incubated with FITC-conjugated IgG2a.
- Figure S2. Translocation of Fab-5.01/CD63 complexes from the cell surface to early endosomes, lysosomes, and MIICs of immature DCs (JPEG file, 860 KB)
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Double labeling to detect internalized Fab-5.01 and EEA1, LAMP-2, and MHC class II molecules was performed. Immature DCs were incubated with 10 µg/mL Fab-5.01 for 1 hour on ice. After washing to remove unbound Fab-5.01, cells were further incubated in culture medium at 37ºC for the indicated periods of time. Fab-5.01 was stained with Cy5-GAM (green in EEA1 panel) or Cy3-RAM (red in LAMP-2 and MHC class II panels). Cells were then incubated with rabbit anti-EEA1 (red; secondary antibody Cy3-GAR), goat anti-LAMP-2 (green; secondary antibody RAG-Cy5), or anti–HLA-DR, -DP, -DQ-FITC labeled (green). Colocalization is visualized by yellow staining. Images are representative of at least four similar experiments. (Original magnification = ×1000, Zoom = 2).
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