Cdk7 cych mat1

k_inact/K_i measurements were determined using kinetic CDK7 enzyme activity data obtained from a peptide substrate mobility shift assay (Blackwell et al., 2009b (link)). Recombinant CDK7/CycH/Mat1 and fluorescent peptide substrate were obtained from Carna Biosciences (CDK7/CycH/Mat1, Cat. 04-108; CTD3 peptide, Cat. 04-108MS). Reactions contained 3-5 nM CDK7 enzyme, 1 μM CTD3 peptide and 1 mM ATP in the assay buffer (20 mM HEPES pH7.5, 0.01% TritonX-100, 5 mM MgCl₂, 0.5 mM DTT). Phosphorylated product and non-phosphorylated substrate were separated and detected using a LabChip EZ Reader® (PerkinElmer) using the following settings: pressure 1.8 psi and voltage differentail −2400V to −500V with separation buffer consisting of 100 mM Hepes pH 7.3, 0.015% Brij-35, 1 mM disodium EDTA, 0.1% coating reagent 3, 5% DMSO and 1X coating reagent 8, similar to previous reports (Tan et al., 2017 (link)) . Nonlinear least-squares regression was performed using the DynaFit software package (Kuzmic, 1996 (link)) using with default settings for a 2-step inactivation model. Figures were generated with GraphPad Prism software (version 6.07).