FIG. 1.

Regulation of HCV NS5A protein expression in UHCV-11 cells and response to IFN with respect to PKR and 2-5A synthetase induction. UHCV-11 cells were seeded in four six-well tissue culture plates at a density of 300,000/well in the presence of tetracycline. After 24 h, they were washed three times with PBS to remove tetracycline and then incubated in culture medium containing different concentrations of tetracycline (1,000, 500, 100, 50, 10, 5, 1, 0.5, or 0 ng/ml [from left to right, lanes 1 to 9]). One set of cells was incubated in the absence of IFN (control [CONT]), and the other set was incubated in the presence of IFN at 500 U/ml. After 18 h of incubation, the cells were lysed in 300 μl of low-salt buffer I (containing 40 mM of NaCl instead of 400 mM) as described in Materials and Methods. The proteins contained in 50 μl of each extract (equivalent to 100,000 cells) were separated by SDS–12.5% PAGE and analyzed by immunoblotting for the presence of NS5A, p69 2-5A synthetase, p100 2-5A synthetase, and PKR.