Fig. 5. Visualization of binding sites for the 42 kDa Fn fragment on the cell surface. Swiss 3T3 vector (A,C) or tTG[2] (A,B,D) transfectants were preincubated in suspension for 1 hour with 25 µg/ml biotinylated 42 kDa fragment, washed and plated on Fn-coated (A) or untreated glass coverslips (B-D). Staining of fixed nonpermeabilized cells is shown. (A) Cell-surface binding sites for the 42 kDa fragment were visualized by double-staining with avidin-rhodamine and anti-ß1 integrin mAb HMß1-1 followed by fluorescein-labeled goat anti-hamster IgG. (B) Codistribution of surface-bound 42 kDa fragment with ß1 integrins, surface tTG and Fn fibrils. Cells were double-stained with avidin-rhodamine and anti-ß1 integrin mAb HMß1-1 or anti-tTG mAbs CUB7402/TG100, followed by secondary fluorescein-labeled IgG (middle and right panels). Alternatively, cells were double-stained with anti-tTG mAbs CUB7402/TG100 and anti-Fn antibody, followed by fluorecein-labeled anti-mouse IgG and Alexa-Fluor 350-labeled anti-rabbit IgG (left panels). (C,D) Cells were stained with avidin-rhodamine to visualize surface-bound 42 kDa fragment and then either stained for Fn (C) or double-stained for Fn and surface tTG (D). (A,B,) Yellow color in merged images shows codistribution of the exogenous 42 kDa fragment with ß1 integrins and surface tTG at focal adhesions (A) or cell-matrix contacts on the dorsal surface (B). (B,D) Arrows point to clusters of surface tTG colocalized with initiation sites of Fn fibril growth (B) or large clusters of Fn fibrils (D). Arrowheads (B) mark codistribution of surface-bound 42 kDa fragment with ß1 integrins and tTG at cell-matrix contacts. Bar, 20 µm (A,B) or 50 µm (C,D).