FIG. 2.

Various levels and timing of viral gene expression in four different cell lines. Serum-starved CCF (permissive), SY5Y (permissive), T98G (semipermissive), and A172 (nonpermissive) cells were infected with HCMV Towne at an MOI of 10 and harvested at the indicated times pi. Equal amounts of cell lysates (from 2 × 10⁵ cells) were used for all lanes in all blots. Abs used are described in Materials and Methods. (A) Comparative levels of viral gene expression in CCF, T98G, and A172 cells. All cell lysates were loaded onto the same blot for the direct comparison of expression levels. An actin loading control was included in all three panels to confirm the loading of equal sample amounts. (B) As protein levels differed dramatically among cell types, optimized concentrations for each primary Ab were determined. Lysates from permissive cells (CCF and SY5Y) were loaded onto one blot, and lysates from semi- and nonpermissive cells (T98G and A172) were loaded onto another. To evaluate IE1 and IE2, UL44, and pp65 in CCF and SY5Y cells, primary Abs were diluted 1:6,000. For the parallel T98G and A172 blots, Abs were diluted 1:2,000 (a threefold difference in Ab concentrations). MCP and pp28 primary Abs were diluted 1:2 for all blots (no differences among cell types). Secondary Ab was used at the same dilution of 1:3,000 for all blots. Blots probed for the same viral protein were exposed using enhanced-chemiluminescence reagents at the same sensitivity levels for identical time periods. (C) Prolonged time course for T98G cells. The infection of T98G cells was extended through 216 h (without passaging), and cells were harvested at the indicated times pi. Primary and secondary Ab concentrations were identical to those given for panel B; however, enhanced-chemiluminescence development times varied slightly, and therefore, slight variations in the results for a given cell type and a particular Ag as presented in the different panels of Fig. 2 may be observed.