FIG. 5.

The anti-VSV P antiserum used in biochemical analyses recognizes all P-protein populations. To rule out the possibility that the observed hypophosphorylation is an artifact generated by an inability of the anti-P antiserum to recognize all available epitopes within the P-protein population, sequential immunoprecipitations was performed. Briefly, anti-P antiserum was added to ³⁵S-cysteine/methionine-radiolabled neuroblastomal lysates from NB41A3 cells treated with IFN-β or vehicle alone. After 24 h, the antigen-antibody complexes were precipitated out of solution using Pansorbin A, taking care to collect the original lysate for subsequent rounds of treatment with anti-P antiserum. After four rounds the original lysate was then treated with an anti-VSV antiserum known to recognize all viral epitopes. The resulting samples were resolved via 12% SDS-PAGE, dehydrated, infiltrated with 2,5-diphenyloxazole, dried, and exposed to film. The anti-P antiserum was able to recognize all epitopes corresponding to the P-protein population in both untreated and IFN-β pretreated NB41A3 cell lysates (No T No V, no treatment, no virus; No T + V, no treatment + virus; IFN-β No V, IFN-β, no virus; IFN-β + V, IFN-β + virus).