FIG 5.
Interaction of amino acid substitution-containing P proteins with N protein. (A) WT P protein and P proteins with amino acid substitutions were coexpressed with N protein in BHK21 cells infected with vTF7-3 prior to transfection. Proteins were metabolically labeled with [35S]methionine-cysteine and then immunoprecipitated (IP) with monospecific polyclonal anti-P antibody. Either immunoprecipitates (top) or total protein samples (bottom) were resolved by SDS-PAGE, followed by autoradiography. (B) Pulse-chase analysis. N protein was expressed in vTF7-3-infected BHK21 cells either alone or with WT P or P226/227A. At 16 h posttransfection, proteins were metabolically labeled with [35S]methionine-cysteine by a 15-min (15′) pulse and then exposed to medium containing excess unlabeled methionine and cysteine for the indicated time periods. Proteins were immunoprecipitated from the soluble fractions with anti-N antibody and resolved by SDS-PAGE (top). Pellet fractions were dissolved in 1× Laemmli sample buffer and resolved by SDS-PAGE (bottom).