FIG. 5.

(A) Increase in IRF-3 phosphorylation during viral infection. Whole-cell extracts were prepared by detergent lysis of HEC-1B cells that were metabolically labeled with [³²P]orthophosphate for a total of 7 h during either mock infection (0 h) (lanes 1 and 2) or infection with NDV for 3 h (lanes 3 and 4) or 6 h (lanes 5 and 6). Extracts were subjected to immunoprecipitation (IP) with a control murine antibody (c) (lanes 1, 3, and 5) or a murine anti–IRF-3 antibody (lanes 2, 4, and 6). Immunoprecipitated proteins were denatured in SDS sample buffer, and 75% of the sample was separated by SDS-PAGE. The gel was fixed, dried, and exposed to film (upper panel). The remaining 25% was analyzed by SDS-PAGE and immunoblotting (IB) with a rabbit polyclonal antibody (α) to IRF-3 (lower panel). The positions of the prestained molecular mass standards are shown in kilodaltons on the left. (B) Effect of protein phosphatase treatment on the migration of IRF-3 in SDS-PAGE. Cytoplasmic (C) and nuclear (N) extracts were prepared from HEC-1B cells that were either untreated (lanes 1 and 2) or infected with NDV (lanes 3 to 7). A total of 100 μg of cytoplasmic extract or 35 μg of nuclear extract was incubated in the absence of phosphatase (lanes 1 to 3), in the presence of PP2A (lanes 4 and 5), or in the presence of PTP1B (PTP) (lanes 6 and 7). The activities of PP2A and PTP1B were inhibited by the inclusion of 10 nM okadaic acid (OA) (lane 5) and 5 mM sodium vanadate (van) (lane 7), respectively. Proteins were separated by SDS-PAGE and detected by immunoblotting (IB) with a murine antibody (α) to IRF-3. (C) Phosphoamino acid analysis of ³²P-labeled IRF-3. Cells were metabolically labeled with [³²P]orthophosphate, and lysates from mock-infected cells (upper panels) or NDV-infected cells (lower panels) were immunoprecipitated (IP) with control (c) antibody (boxes 1 and 3) or antibody to IRF-3 (boxes 2 and 4). Following SDS-PAGE, proteins were electroblotted to Immobilon-P membranes, and the area containing IRF-3 or the adjacent area from control immunoprecipitations was subjected to partial acid hydrolysis. The hydrolysates were resolved by two-dimensional electrophoresis on a TLC plate. Positions of the serine, threonine, and tyrosine phosphoamino acid internal standards are indicated by dotted circles (pS, pT, and pY, respectively), and origins are denoted by a plus sign.