FIG. 1.

Characterization of ISG expression following tetracycline (Tet) induction in FLP-IN/ISG cell lines. (A) Demonstration of the desired ISG expression in the seven newly established cell lines. FLP-IN T Rex-derived cell lines that inducibly express individual ISGs were established as described previously (21). The cells were cultured in the absence or presence of tetracycline for 48 h and then harvested. The levels of N-terminally FLAG-tagged ISG protein expression in cell lysates were determined by Western blot analysis with a monoclonal antibody against the FLAG tag as previously described (21). M. W., molecular mass. (B) Comparison of the kinetics and levels of ISG expression upon IFN-α treatment and tetracycline induction. Huh7, HeLa, and parental FLP-IN T Rex cells were left untreated or treated with 1,000 IU/ml IFN-α, and cells were harvested at the indicated times after IFN-α treatment. Three FLP-IN/ISG cell lines that express MxA, PKR, and ISG15, respectively, were cultured in the absence or presence of 1 μg/ml tetracycline, and cells were harvested at the indicated times after the addition of the antibiotic. The levels of the three IFN-induced proteins in cell lysates were determined by Western blot analysis. Briefly, cell monolayers were washed once with phosphate-buffered saline and lysed with 1× Laemmli buffer. A fraction of the cell lysate was separated on SDS-12% polyacrylamide gels and electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with phosphate-buffered saline containing 5% nonfat dry milk and probed with antibodies against MxA (Proteintech Group), PKR (a gift from Pat Romano, Institute for Hepatitis Virus Research, Hepatitis B Foundation, Doylestown, PA), ISG15 (Cell Signaling), and β-actin (Chemicon International). Bound antibody was revealed by IRDye secondary antibodies and visualized by the Li-COR Odyssey system.