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. Author manuscript; available in PMC: 2010 Jun 1.
Published in final edited form as: Dev Dyn. 2009 Jun;238(6):1346–1357. doi: 10.1002/dvdy.21920

Figure 4.

Figure 4

Transient whole embryo reporter assays for Mix.3 trancriptional activation activity. Embryos were injected at the one-cell stage with a Mix-reporter construct (100 pg; P3luc = three tandem Mix-binding sites cloned upstream of a minimal TATA-box driving luciferase expression) and combinations of synthetic mRNA encoding Mix.3, a deletion mutant of Mix.3 that lacks the CDK9 interaction domain (M3Δ123), CDK9, cyclin T2 and cyclin K. The injection cocktail also included a thymidine kinase promoter-Renilla plasmid (TK-Renilla; 50 pg) as an internal control. Luciferase and Renilla activities were measured in three pools of ten embryos each at stage 11. CDK9/cyclin T2, but not CDK9/cyclin K, co-injection blocks Mix.3 transactivation of the P3luc reporter. The inhibition of Mix.3 activity on the P3luc reporter is dependent on the interaction of Mix.3 with CDK9 as deletion of the CDK9-interaction domain from the carboxyl terminus of Mix.3 alleviated the inhibition of CDK9/cyclin T2 (sample Mix.3D123/CDK9/cyclinT2). Mix.3/Cyclin T2, without added CDK9, showed no significant change in P3luc activity relative to Mix.3 mRNA injection alone.