Fig. 8. Intracellular presence of peptide IFNGR-1(253-287) inhibits binding to IFNGR of extracellular IFN and subsequent activation of STAT1. (A) Presence of extracellular peptide IFNGR-1(253-287) did not inhibit binding of ¹²⁵I-IFN to P388D1 cells at the concentrations to be used in subsequent experiments. Unlabeled murine IFN or peptide IFNGR-1(253-287), as indicated, was added at a final concentration of 1 µM to P388D1 cells at 4°C along with 10 nM of ¹²⁵I-IFN, and cells were incubated at 4°C for 30 minutes. Control cells were incubated with ¹²⁵I-IFN in the absence of any competitor. Cells were then washed and bound IFN determined. Samples were run in triplicate and values plotted as mean±s.d. (B) Intracellular accumulation of peptide IFNGR-1(253-287) in P388D1 cells by pinocytosis was accomplished by incubating cells with either 25 µM (lane 2) or 50 µM (lane 3) of peptide at 37°C for 1 hour. Cells used in lanes 1 and 4 did not receive any peptide. Cells were then washed at room temperature to remove extracellular peptide and then incubated with ¹²⁵I-IFN (10 nM) along with 1 µM of IFNGR-1 peptide for 5 minutes at 37°C. Control cells (lane 1) were washed in ice-cold medium and then incubated with ¹²⁵I-IFN at 4°C without peptide. After ¹²⁵I-IFN incubation, all cells were washed at 4°C and then acid-washed at 4°C to remove surface-bound ¹²⁵I-IFN. Cells were then lysed and immunoprecipitated with antibodies to IFNGR-1. After western transfer of immunoprecipitates to nitrocellulose membranes, ¹²⁵I-IFN associated with IFNGR-1 was detected by autoradiography. Total IFNGR-1 immunoprecipitated was followed by immunodetection with IFNGR-1 antibodies (lower panel). (C) Conditions are the same as in (B), except that lysates were immunoprecipitated with STAT1 antibodies and tyrosine phosphorylation of immunoprecipitated STAT1 was followed by immunodetection with antibodies specific for Tyr701-phosphorylated STAT1. Total immunoprecipitated STAT1 was followed by reprobing blots with antibodies to STAT1 (lower panel).