Noncanonical IFN Signaling: Mechanistic Linkage of Genetic and Epigenetic Events

Figure 2

Intracellular presence of peptide IFNGR-1(253-287) inhibits binding to IFNGR of extracellular IFNγ and subsequent activation of STAT1α. (a) Presence of extracellular peptide IFNGR-1(253-287) did not inhibit binding of ¹²⁵I-IFNγ to P388D1 cells at the concentrations to be used in subsequent experiments. Unlabeled murine IFNγ 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-IFNγ, and cells were incubated at 4°C for 30 minutes. Control cells were incubated with ¹²⁵I-IFNγ in the absence of any competitor. Cells were then washed and bound IFNγ 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-IFNγ (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-IFNγ at 4°C without peptide. After ¹²⁵I-IFNγ incubation, all cells were washed at 4°C and then acid-washed at 4°C to remove surface-bound ¹²⁵I-IFNγ. Cells were then lysed and immunoprecipitated with antibodies to IFNGR-1. After Western transfer of immunoprecipitates to nitrocellulose membranes, ¹²⁵I-IFNγ 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 STAT1α antibodies and tyrosine phosphorylation of immunoprecipitated STAT1α was followed by immunodetection with antibodies specific for Tyr701-phosphorylated STAT1α. Total immunoprecipitated STAT1α was followed by reprobing blots with antibodies to STAT1α (lower panel).

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