FIG. 4.

Stability of NS1 proteins and their interaction with chicken CPSF. (A) Analysis of NS1 expression by pulse-chase radioactive labeling and immunoprecipitation. The SW/FJ/01 and SW/FJ/03 NS1 proteins were expressed and labeled as described in Materials and Methods. The cells were lysed after being pulse labeled or at different chase time points, and cell lysates were immunoprecipitated with antibodies against NS1, followed by SDS-PAGE analysis. Lane 1, control (empty plasmid DNA vector-transfected cells); lanes 2 to 4, pBlue-NS1-01-transfected samples; lanes 5 to 7, pBlue-NS1-03-transfected samples. Lanes 2 and 5, samples prepared after being pulse labeled; lanes 3 and 6, samples prepared after a 4-h chase; lanes 4 and 7, samples prepared after a 7-h chase. The arrow indicates the NS1 protein band. (B) Analysis of CPSF binding. HA-tagged NS1 constructs were expressed by in vitro translation. Flag-tagged CPSF was expressed in transfected 293T cells. The cell lysate was mixed with the NS1 proteins and subjected to coimmunoprecipitation (IP) using a monoclonal anti-Flag antibody coupled to protein A-Sepharose. The precipitated proteins were analyzed for the HA-tagged NS1 protein and Flag-CPSF by Western blot analysis using HA tag- and Flag-specific monoclonal antibodies, respectively. Lane 1, SW/FJ/01 NS1; lane 2, SW/ FJ/03 NS1.