Figure 9.

Analyses of CDK7 and pUL97 activities applying in vitro kinase assays. FLAG-tagged pUL97 and its inactive mutant pUL97(K355M)–FLAG (KM) were transiently transfected in 293T cells and harvested 48 h post-transfection (A); HFFs were infected with HCMV or remained uninfected (mock) and harvested 120 h post-infection (B). Total cell lysates were subjected to CoIP and IVKA. Two different NaCl concentrations were used in the CoIP buffer (A, 500 mm NaCl; B, 150 mm NaCl) to achieve optimized stringency of CoIP. Each IVKA reaction was supplemented by addition of either CDK7–cyclin-H–MAT1 complex (200 ng), Rb-CTF (1 μg), CDK7 inhibitor LDC4297 (1 μm), or pUL97 inhibitor MBV (3 μm) as indicated. Upper panel, IVKA (autoradiogram) and detection of CDK7, cyclin H, and MAT1 on the IVKA membrane (Western blot staining); lower panels, total input levels of different proteins as expression control. Note that Western blot splicing was performed to integrate the relevant lanes as indicated by vertical marking lines.