Fig. 6.

Human CDK7 does not increase pUL97 in vitro kinase activity. (A) 293T cells were transiently transfected with plasmids encoding pUL97-Flag. Cells were lysed 2 d post-transfection, and pUL97-Flag and CDK7 were immunoprecipitated using the indicated antibodies; a mouse Fc fragment served as a negative control. Dynabeads-bound proteins were eluted in 150 µl enzyme buffer. 25 µl of eluted Dynabeads were denatured in 25 µl 2x loading buffer and subjected to SDS-PAGE and Wb to confirm successful precipitation of pUL97-Flag and CDK7. (B) Samples derived from immunoprecipitation of CDK7 plus pUL97-Flag, as well as Fc control samples, were subjected to a qSox-IVKA (using the pUL97-specific sensor peptide AQT0258). Optionally, 0.01 µM of CDK7-specific inhibitor LDC4297 (corresponds to the mean antiviral EC₅₀ value) or equal amounts of DMSO were added to the reactions. Background activity determined with the Fc control was subtracted from values of combined pUL97-Flag plus CDK7 kinase activity. Mean values ± SD derived from one representative experiment are given, as derived from measurements in triplicates. Statistical analysis was performed using an ordinary two-way ANOVA: n.s., not significant.