FIGURE 2.
Mapping IFNAR1 domains critical for interaction with SOCS1. A, the histogram shows relative expression of an ISRE-luciferase reporter in IFNAR1−/− MEFs transiently cotransfected with either mu-IFNAR1, IFNAR1Y445F, IFNAR1Y518F, IFNAR1Y529F, IFNAR1Y576F, IFNAR1Y518F/Y529F/Y576F(3F), IFNAR1Y445F/Y518F/Y529F/Y576F(4F) IFNAR1Δ511, or IFNAR1Δ471 (represented diagrammatically in the lower panel) with or without mu-SOCS1. Y denotes tyrosine residues. Cells were stimulated with 50 IU/ml mu-IFNα for 7 h prior to luciferase readings. Luciferase activity is normalized against a TK-Renilla reporter to control for transfection efficiency and is expressed relative to wild-type IFNAR1 in the absence of SOCS1, which is denoted as 100%. Data are represented as mean ± S.E. of three experiments. ***, p < 0.001. B, immunoprecipitation (IP) Western blot analyses (WB) of IFNAR1 mutants and SOCS1. IFNAR1, IFNAR1(4F), IFNAR1Δ 511, or IFNAR1Δ471 were cotransfected with SOCS1-FLAG into HEK293T cells. Following expression, immunoprecipitation of IFNAR1 or the IFNAR1 mutants was performed using anti-IFNAR1 antibody. Immunoprecipitates and whole cell lysates (WCL) were separated by SDS-PAGE and immunoblotted with anti-FLAG to detect SOCS1-FLAG. Blots are representative of triplicate experiments.