FIG. 5.

Effect of proteinase K digest on cellular proteins and quantification of protease-resistant HCV nonstructural proteins in CRCs. Equal amounts of CRCs prepared from replicon cell clone 9-13 were incubated for 60 min at 25°C in the presence or absence of 1% Triton X-100 and/or 0.8 (+) or 8 (++) mg of proteinase K/ml and/or 0.2 (+) or 2 (++) U of S7 nuclease/μl, respectively, as indicated above each lane. The reaction was stopped by the addition of 1.4 mM PMSF and 2.75 mM EGTA and boiled in sample buffer, and total protein equivalent to 2 μl of the CRCs was subjected to SDS-10% PAGE. In the fivefold-concentrated samples, the proteins were trichloroacetic acid precipitated, and the equivalent of 10 μl of CRCs was loaded. Proteins were either visualized by silver staining (A) or subjected to immunoblot analysis (B) with monoclonal antibodies specific for the ER luminal part of calnexin (upper panel), a polyclonal antiserum raised against HCV NS3 or NS4B (middle two panels), and monoclonal antibodies specific for HCV NS5B (bottom panel), as indicated at the right. The concentrated fractions (lane 12) are shown from identical expositions of the same blot. A serial dilution of purified NS5B was loaded in parallel for quantification as depicted at the bottom of the figure.