Fig. 2.

Time course of dissociation of the radiolabeled nonselective Bn analog agonist (Peptide-1) and the BRS-3 peptide antagonist Bantag-1 from hBRS-3/BALB 3T3 cell membranes (top) or intact cells (bottom). hBRS-3/BALB (1 × 10⁶ cells/ml) were incubated for 60 minutes at 37°C with 50 pM ¹²⁵I-labeled ligand (¹²⁵I-Bantag-1 or ¹²⁵I-Peptide-1) and with or without unlabeled 1 μM peptide-1 in binding buffer. Membranes were incubated with 50 pM radioligand (i.e., ¹²⁵I-Bantag-1 or ¹²⁵I-Peptide-1) at room temperature for 60 minutes with or without unlabeled 1 μM peptide-1 ligand in binding buffer. Then a final concentration of 1 μM peptide-1 was added, and the membranes or cells were incubated at room temperature or 37°C, respectively. At various times, 100-µl aliquots were taken and saturable binding determined as described in Methods. In each experiment, each value was determined in duplicate, and results are from at least four separate experiments.