Fig. 1. Kinetics and localization of III_1c binding to fibroblast monolayers. Human foreskin fibroblasts were grown to 90% confluence over three days in complete medium. (A) The growth medium was removed and replaced with fresh medium containing 5% fibronectin-depleted serum and 20 µM various His-tagged fibronectin modules for the indicated times: bound modules were detected by an ELISA using an antibody against the His tag. (B) Cell layers were incubated with 20 µM of III_1c for the indicated times and bound III_1c was detected by indirect immunofluorescence using an anti-His-tag (HIS) antibody. III_1c was then visualized using a secondary antibody containing Alexa Fluor-594. Cells were costained for fibronectin (FN) using a polyclonal antibody against the entire molecule and a secondary antibody tagged with Alexa Fluor 488. Overlays of both images are shown in yellow. (C) Increasing concentrations of FN modules were incubated with the cell layers for 2 hours. Cell layers were washed and fixed, and bound protein was measured as described in A. In A and C, each point represents the average of triplicate samples. Error bars indicate standard error of the means. The data represents one of four experiments performed.