Fig. 3. Specific binding of GFa and CPp to vß5 integrin. (A) ¹²⁵I-CPp was incubated with aliquots of HEK-293 cells (as described in the legend to Fig. 2B) that had been preincubated with the indicated antibody dilution for 1 hour at 4°C. When indicated, ¹²⁵I-CPp was preincubated either with purified vß5 integrin (250 or 500 ng) or 1ß1 integrin (250 ng) in a total volume of 0.2 ml for 1 hour prior to radioreceptor-binding assay. The white bar (furthest right) refers to the radioactivity specifically bound to HEK-293-v cells, which had been exposed to ¹²⁵I-CPp for 3 hours at 4°C. Non-specific binding was assessed by including additional duplicate samples containing unlabeled 10 nM CPp. Specific binding to HEK-293 cells was taken as 100% and the extent of inhibition by anti-integrin antibodies or by vß5 integrin or 1ß1 integrin was calculated relative to this value. Values represent the mean ± s.d. of two independent experiments performed in duplicate. ^*P<0.005; ^**P<0.0006. (B) 24-well cell culture dishes were coated with 1 mg/ml BSA or 25 µg/ml vitronectin or 100 pM GFa overnight at 4°C and extensively washed with 1x PBS. For each sample, 10⁵ HEK-293 cells were preincubated with the indicated dilution of antibodies or non-treated (none) in a final volume of 0.3 ml for 1 hour and allowed to adhere in an 1-hour-assay at 37°C. The number of adherent cells was counted and is given as the percentage of the total cell population, representing the average of three different experiments performed in duplicate. ^*P<0.0001 compared with GFa. (C) 6% SDS-PAGE followed by western blotting with anti-v integrin polyclonal antibody. 15 µl of protein A-anti-uPA antibody Sepharose with (+ GFa) or without (- GFa) 5 µg of GFa was incubated for 2 hours at 25°C in a total volume of 60 µl, washed and further incubated with 500 ng of vß5 integrin. Sepharose-bound proteins were isolated by centrifugation and the supernatants (Sup) and the eluates (El) were separated by 7.5% SDS-PAGE. 100 ng of purified vß5 integrin protein was loaded as a control.