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. Author manuscript; available in PMC: 2015 Jan 31.
Published in final edited form as: Nature. 2014 Jun 22;511(7511):616–620. doi: 10.1038/nature13393

Extended Data Figure 1. THZ1 demonstrates time-dependent inhibition of CDK7 in vitro and covalent binding of intracellular CDK7.

Extended Data Figure 1

a, THZ1 but not THZ1-R shows time-dependent inhibition. LanthaScreen® Eu Kinase Binding assay was conducted at Life Technologies in a time-dependent manner (20, 60, and 180 min.) showing that THZ1 but not THZ1-R shows time-dependent inhibition of CDK7. b and c, Pre-incubation of THZ1 increases CDK7 inhibitory activity in vitro. Recombinant CAK complex was incubated with THZ1 (b) or THZ1-R (c) in dose response format with or without pre-incubation prior to ATP (25 µM) addition. Kinase reaction was then allowed to proceed for 45 minutes at 30°C. d, Workflow of bio-THZ1 pull down competition experiment. e, bio-THZ1 pulls down CDK7 from cellular lysates. Loucy cellular lysates were incubated with bio-THZ1 (1 µM) with or without THZ1 (10 µM) and streptavidin-precipitated proteins were probed for CDK7. IB = immunoblot. f, Free intracellular THZ1 competes in a dose-dependent manner for bio-THZ1 binding to CDK7. Loucy cells were treated with increasing concentrations of THZ1 or with 10 µM THZ1-R for 4 hrs. Cellular lysates were incubated with bio-THZ1 and processed as indicated in a. g, bio-THZ1 labels CDK7 in lysates. Loucy cellular lysates were incubated with bio-THZ1 at 4°C for 12 hrs followed by immunoprecipitation of CDK7 at 4°C for 3 hrs. Precipitated proteins were washed and probed with streptavidin-HRP.