Figure 5.

Mouse embryonic fibroblasts expressing Endo180ΔEx2–6 have a defect in collagen binding and uptake. Mouse embryonic fibroblasts (MEFs) were seeded into 35-mm dishes for 24 h. The cells were then incubated with OregonGreen488 (OG)–collagen-IV or OG–gelatin for 2 h. (A) Cells were analysed by flow cytometry. +/+ cells are indicated by red lines, +/ΔEx2–6 cells by green lines and ΔEx2–6/ΔEx2–6 cells by blue lines. In the top two graphs, black lines indicate the binding of FITC (fluorescein isothiocyanate)–BSA to +/+ cells. In parallel (bottom two graphs), cells were stained with an anti-integrin β₁ monoclonal antibody followed by Alexa488 anti-rat Ig, or with biotinylated anti-integrin β₃ followed by phycoerythrin–streptavidin, and were analysed by fluorescence-activated cell sorting (FACS). Black lines indicate binding of Alexa488 anti-rat Ig and PE-streptavidin alone to +/+ cells. (B) Cells were collagenase treated to remove surface-bound collagen, fixed, permeabilized and stained with anti-Endo180 followed by Alexa555 anti-rabbit Ig; nuclei were counterstained with TO-PRO-3. Scale bar, 20 µm.