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. 2008 Aug 22;283(34):23169–23178. doi: 10.1074/jbc.M802729200

FIGURE 2.

FIGURE 2.

RIG-I and MAVS complex with NIK. A, HEK293 cells were transfected with eukaryotic expression vectors encoding Myc-tagged NIK (Myc_ NIK) and different deletion mutants of FLAG epitope-tagged RIG-I, including full-length RIG-I (RIG), the NH2-terminal CARD domains (RIG_N), and the COOH-terminal helicase domain (RIG_C). 36 h after transfection, whole cell lysates were prepared and Myc_NIK immunoprecipitated (IP) using anti-Myc Ab. RIG-I association were detected by Western immunoblot (WB) probing with anti-FLAG Ab. Black arrowhead shows the location of full-length of RIG-I; white arrowhead indicates RIG_N (the first panel from top). To monitor IP recovery, the presence of Myc_NIK in the immunoprecipitate was measured by probing the same membrane with anti-Myc Ab (second panel from top). Conversely, Flag_RIG and its deletion mutants were immunoprecipitated using anti-FLAG Ab, and Western immunoblot was performed using anti-Myc Ab (third panel from top). Note that Myc-NIK is only seen in the IPs from RIG and RIG_N co-transfected cells. To monitor IP recovery, the presence of RIG-I was measured by anti-FLAG Ab; each isoform is indicated by black arrowheads (bottom panel). B, HEK293 cells were transfected with Flag_GFP_NIK and different forms of Myc_MAVS. IP was conducted using anti-FLAG and MAVS detected by anti-Myc Ab in a Western blot (top panel). IP of Flag_GF-P_NIK was confirmed by Western blot (WB) (second panel). Conversely, the MAVS was immunoprecipitated using anti-Myc Ab, and the presence of NIK was determined in Western blot using anti-FLAG Ab (third panel). IP of Myc-MAVS was confirmed by reprobing the membrane with anti-Myc (bottom panel). Note that only full-length MAVS associates with NIK. C, nondenaturing coimmunoprecipitation of RSV-infected cell extracts using anti-IgG (lane 1) or anti-MAVS Ab (lane 2). Immune complexes were washed, and the presence of NIK determined by Western immunoblot. Reactivity of anti-MAVS Ab was determined by probing cellular lysates (lane 3), where full-length MAVS and its characteristic alternative translational initiation forms are seen (compare B, lane 1). A specific band of 150 kDa was observed only in the MAVS immunoprecipitates. NS, nonspecific.