Fig. 2. Binding of PGI/AMF to dimeric FN at neutral and acid pH. The fluorescent signal due to binding of PGI/AMF-FITC to uncoated wells of a 96-well plate, to wells coated with 20 µg ml^–1 BSA or FN, or to wells plated with NIH-3T3 cells was amplified by anti-FITC and Alexa488 anti-rabbit secondary antibodies and measured with a fluorescence plate reader (A). Absolute relative fluorescence values were normalized to maximal values and binding at pH 5 and pH 7.5 in the presence or absence of PGI/AMF-FITC was determined. To assess relative FN levels in the wells containing soluble FN or NIH-3T3 cells, parallel wells were labeled with anti-FN and Alexa488 anti-rabbit secondary antibodies (±s.e.m., n=4). Alternatively, NIH-3T3 cells were plated for 2 days on cover slips coated with 20 µg ml^–1 of FN. The cells were then incubated with 25 µg ml^–1 bPGI/AMF for 60 minutes at 37°C. After fixation with 3% paraformaldehyde, bPGI/AMF was revealed with Texas Red-streptavidin (B) and FN labeled with mouse anti-FN mAb and Alexa-488 conjugated anti-mouse secondary antibody (C). bPGI/AMF appears in red and FN in green and co-localization of the two appears in yellow in the merged image (D). PGI/AMF binds selectively to the fibrillar form of FN in NIH-3T3 cells plated on a FN substrate. Bar, 20 µm.