FIG. 5.

MAD78 regulation of JAK-STAT signaling is attenuated. (A) A549 cells were mock infected (lanes 1 to 3) or infected (MOI = 5) with WNV (TX02, lanes 4 to 6; MAD78, lanes 7 to 9). Twenty-four h postinfection, cells were pulse treated with 1,000 U IFN-α for 0, 15, or 30 min, and whole-cell lysates were collected and analyzed by immunoblotting to determine WNV protein (NS3) abundance or the abundance of the active, tyrosine-phosphorylated (P) isoforms of Tyk2, STAT1, and STAT2. (B) A549 cells were mock infected or infected with TX02 or MAD78. Cells were then treated with 1,000 U IFN-α for the times indicated. Proteins were immunoprecipitated from whole-cell lysates by use of an antiphosphotyrosine antibody, and JAK1 immunoblot analysis was performed subsequently. (C) A549 cells were infected with TX02 or MAD78 and treated with IFN as described for panel A. Whole-cell lysates were analyzed for the presence of phospho-JAK1. (D) A549 cells were mock infected (M; lanes 1 and 12) or infected (MOI = 5) with TX02 (lanes 2 to 11) or MAD78 (lanes 13 to 22). In 5-h increments, cells were pulse treated with 1,000 U IFN-α for 30 min, and whole-cell lysates were collected and analyzed by immunoblotting to detect GAPDH, WNV, phosphotyrosine STAT isoforms, and total STAT1 or STAT2 abundance. Bars at left indicate the positions of molecular mass (kilodalton) standards. Arrows at right denote the positions of the indicated WNV proteins.