Fig. 1: Generation of an Analog-Sensitive (AS) version of CDK16. | Experimental & Molecular Medicine

Fig. 1: Generation of an Analog-Sensitive (AS) version of CDK16.

From: Phosphoregulation of the oncogenic protein regulator of cytokinesis 1 (PRC1) by the atypical CDK16/CCNY complex

Fig. 1

a CDK16 phosphorylates CCNY in vitro. ATPγS was used as a phosphodonor, and thiophosphorylation (ThioP) was evaluated by western blotting. CDK16 and CCNY were purified from E. coli. b Identification of the gatekeeper residue in the ATP-binding pocket by sequence alignment with other CDKs. The motif for this conserved sequence was generated using WebLogo. c In vitro kinase assay using WT- or AS-GST-CDK16 with GST-CCNY as a substrate. The indicated N6-substituted ATPγS analog was used as a phosphodonor. d In vitro kinase assay using WT- or AS-GST-CDK16 with ATPγS or N6-Ph-ATPγS, respectively, in the presence of different inhibitors (1–5) to test the ability of these inhibitors to inhibit the AS mutant. e In vitro kinase assay conducted to establish the specific IC50 of 3MBPP1. WT- or AS-GST-CDK16 was incubated with [γ-32P]-ATP in the presence of different concentrations of 3MBPP1 for the indicated time intervals (in min)

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