FIG. 7.

Two-dimensional electrophoresis of VSV proteins from cell lysates demonstrates pI shifts consistent with hypo- and hyperphosphorylation. (A) Neuroblastomas treated with and without 400 U/mL IFN-β were infected with VSV (MOI = 3), then labeled with ³⁵S-cysteine/methionine (100 μCi/mL). Cell lysates were subsequently immunoprecipitated with anti-VSV antiserum (at 6 hpi), and resolved via isoelectric focusing on pH 3–10 ampholyte gradients. The resulting isoelectrically-focused samples were further resolved via 14% SDS-PAGE. These gels were then dehydrated in DMSO, infiltrated with 2,5-diphenyloxazole, dried, and exposed to film. The autoradiographs were then subjected to densitometric analysis using Un-Scan-It Gel software version 5.1. (B) A shift in the P-protein population towards a more basic distribution was observed in cell lysates isolated from IFN-β-pretreated neuroblastomas, compared to untreated control cells, consistent with hypophosphorylation. (C) A shift in the peak M-protein population towards a more diffuse acidic distribution was observed in cell lysates isolated from IFN-β-pretreated neuroblastomas, compared to untreated control cells, consistent with hyperphosphorylation. (D) The G protein from mock-treated NB41A3 cells focused into two broad acidic populations between pH 3 and 5. However, in cellular lysates from IFN-β-treated cells these two protein populations condense into one broad acidic population centered around pH 3. The asterisk represents each proteins control band for conventional SDS-PAGE. The dagger represents a background, nonviral protein that was verified by running the uninfected control samples (data not shown). Autoradiographs and densitometry results shown are representative of experiments done in triplicate (No T + V, no treatment + virus; IFN-β + V, IFN-β + virus).