Fig. 4. Matrix incorporation of exogenous fibronectin on 2D and 3D fibronectin substrates. (A) CHO(B2)5 cells were plated on 2D and 3D substrates in the presence of 10 µg/ml rat pFN. Fibronectin levels in DOC-insoluble matrix were detected with IC3 anti-rat fibronectin antibody at the indicated times. Exogenous fibronectin was also incubated with 3D matrix in the absence of cells and background binding was analyzed at 23 hours (far right-hand lane). (B) 3x10⁵ CHO(B2)5 cells were plated on substrates with 10 µg/ml rat pFN with or without 125 µg/ml 70 kDa fibronectin fragment. DOC-insoluble samples were collected and analyzed by immunoblotting after 7 hours incubation. The right lane shows no background fibronectin binding in the absence of cells. (C) CHO(B2)5 cells plated with 4 µg/ml exogenous rat pFN on 3D (left) and 2D (right) substrates for 15 hours were fixed and stained with IC3 antibody and fluorescein-goat anti-mouse IgG plus rhodamine-conjugated phalloidin. Bars, 20 µm.