Figure 1.

Protein expression and activity of 2′5′AS in non-stimulated and IFN-α stimulated beta TC3 and alpha TC3 cells. After 24 hours of IFN-α stimulation, cell lysates were prepared. (a) For immunoblot, cell extracts were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The polyclonal antibody specific to human 2′5′AS (cross-reactive with murine 2′5′AS) was used to detect the expressed proteins. The polyclonal antibody against mouse GAPDH was used to quantitate the loading amounts; (b) For assay of 2′5′AS activity, lysates were incubated with 0.15 μCi α ³²P-ATP for 120 minutes at 30°C in the presence of 100 μg/ml poly(I:C), and then separated by ascending TLC. The generated 2′5′-oligoadenylates (2′5′A) were quantitated by PhosphorImager analysis. Enzyme activity was expressed in units per milligram protein (n = 3).