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. 2009 Dec;22(6):353–369. doi: 10.1089/vim.2009.0057

FIG. 6.

FIG. 6.

VSV M protein is hyperphosphorylated in both neuronal cell lysates and virions derived from cells pretreated with IFN-β. (A) Analysis of 32P and 35S metabolically incorporated into M protein from dual-labeled cell lysates. As previously described in Fig. 3, neuroblastomas were treated with 400 U/mL IFN-β or vehicle alone, infected with VSV (MOI = 3), and dual-labeled with 32P-orthophosphate (50 μCi/mL) and 35S-cysteine/methione (100 μCi/mL). Cell lysates were subsequently immunoprecipitated with anti-VSV antiserum (at 6 hpi), resolved via 12% SDS-PAGE, stained with Coomassie Brilliant Blue, and exposed to film. Bands corresponding to VSV M protein were excised, using the developed film as a guide, dissolved in 30% H2O2, and added to scintillation fluid (1:6) for counting. The 35S:32P ratio associated with the M protein decreased in neuronal lysates derived from IFN-β-pretreated cells, representative of hyperphosphorylation. (B) Analysis of 32P and 35S metabolically incorporated into M protein from virions. Viral supernatants were collected and viral lysates were prepared as described in Fig. 4. Viral lysates were then resolved via 12% SDS-PAGE, stained with Coomassie Brilliant Blue, and exposed to film. Bands corresponding to virion-associated M protein were excised, using the resulting autoradiograph as a guide, then dissolved in 30% H2O2, and added to scintillation fluid (1:6) for counting. The 35S:32P ratio from virion-associated M protein decreased in virus derived from IFN-β-pretreated cells, representative of the hyperphosphorylation observed within the cell lysates. Analogous experimentation could not be performed with (C) NIH/3T3 and (D) L929 cell lines, due to undetectable levels of VSV M in cell lysates (*) and the corresponding supernatants (*) (supernatant 35S:32P ratio data not shown). Error bars correspond to 95% CI and p-values were calculated based on Satterthwaite's method for calculating t-values from independent samples of unequal variances (n = 12) (No T + V, no treatment + virus; IFN-β + V, IFN-β + virus).