FIG. 3.
Analysis of structural protein expression in cell lines carrying sfl genomes. (A) Detection of HCV core protein by Western blotting. Lysates, each corresponding to 2 × 105 cells of the given cell lines, were loaded onto a gel, separated by electrophoreses, blotted, and probed with an HCV core-specific monoclonal antibody. (B) Pulse-chase and endoglycosidase H analysis of HCV glycoprotein complexes. Cells were incubated with [35S]methionine/cysteine-containing medium for 1 h, and after the cells were washed extensively, nonradioactive medium was added for the times specified above the lanes. Glycoprotein complexes were isolated by immunoprecipitations from cell lysates under nondenaturing conditions and were either mock treated (− EndoH) or deglycosylated by endoglycosidase H digestion (+ EndoH). After SDS-PAGE, proteins were detected by autoradiography. The deglycosylated forms of the proteins are labeled with asterisks. (C) Analysis of HCV glycoprotein complexes under reducing and nonreducing conditions. Immunoprecipitations were performed in the presence of 20 mM iodoacetamide in the lysis and wash buffer. Prior to loading, half of each sample was boiled in denaturing buffer with or without 2-ME as indicated. Proteins were detected as described above.