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. 2012 May;86(10):5742–5751. doi: 10.1128/JVI.07006-11

Fig 5.

Fig 5

Exogenous FLAG-CDK8 kinase activity is regulated by PP2A and RV-cyclin. HeLa cell nuclear extracts with transiently expressed FLAG-CDK8, FLAG-CDK8 with RV-cyclin (RV-cyclin), FLAG-CDK8 with cyclin C (cyclin C), or kinase-deficient FLAG-CDK8 (FLAG-CDK8 KD) were immune precipitated with anti-FLAG antibody or with combined anti-FLAG and anti-PP2A (αPP2A) antibodies for kinase assay with GST-CTD as the substrate. Equal aliquots of washed Ab-protein G magnetic beads were suspended in a common reaction mix with or without added OA (10 nM). After incubation, GST fusion substrate was captured with added glutathione magnetic beads. IP and substrate were washed and separated by polyacrylamide gel electrophoresis and blotted onto nylon for the imaging of labeled GST-CTD substrate (32P-CTD). The blot was subsequently probed with specific antibodies for the CTD (74 to 76 kDa; αCTD) and FLAG-CDK8 (56 kDa; αFLAG). The chart presents signal (relative band volume) from the phosphorimage of the CTD band.