FIG. 8.

Roscovitine treatment affects steady-state levels of endogenous cdk9 and not those of overexpressed cdk9HA. (A) The G₀-synchronized cdk9HA-overexpressing HFF were released into G₁, HCMV infected at an MOI of 5, and seeded onto coverslips. At 12 h p.i., the cells were washed with PBS, fixed in formaldehyde, permeabilized, and immunostained with IE2 (IgG1) and HA (IgG2a). As a control, as second anti-HA antibody (rabbit polyclonal) was used to confirm specific staining (not shown). Specific antibody staining was detected with fluorescein isothiocyanate- or tetramethyl rhodamine isocyanate-conjugated isotype-specific secondary antibodies. Nuclei were stained with Hoechst dye. The white arrow in each panel indicates a region of colocalization, although more are present. All of the images are confocal optical sections of 0.2 μm at a magnification of ×1,000 under oil immersion. (B and C) The immunocomplexes from mock cells containing the exogenously expressed cdk9 were assayed for kinase activity towards purified GST-CTD (B), and the corresponding immunoprecipitates were run on an 8% SDS-polyacrylamide gel and subjected to Western blot with an antibody specific for cdk9 (C). Pre, preimmunoprecipitate; IP, immunoprecipitate; Post, postimmunoprecipitate. (D) The G₀-synchronized cdk9HA-overexpressing HFF and untransduced HFF were HCMV infected at an MOI of 5 or mock infected in the presence of 16 μM roscovitine or DMSO. Samples were collected at 8 and 24 h p.i. Lysates consisting of an equal number of cells were loaded on a 10% SDS-polyacrylamide gel. Western blot analysis was performed as described above for cdk9. M, mock treated; V, HCMV infected; −, DMSO treated; +, roscovitine treated at the time of infection; F, untransduced HFF cells; 9, cdk9HA-overexpressing cells; *, endogenous cdk9; **, cdk9HA.