XL413 monohydrochloride

20 ng of purified human DDK is pre-incubated with increasing concentrations of each DDK inhibitor for 5 min. Then 10 µCi (γ)-³²P ATP and 1.5 µM cold ATP are added in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, and 1 mM DTT and incubated for 30 min at 30°C. The proteins are denatured in 1X Laemmli buffer at 100°C followed by SDS-PAGE and autoradiography on HyBlot CL film. Auto-phosphorylation of DDK is used as an indicator of its kinase activity. ³²P-labeled bands are quantified using ImageJ and the IC₅₀ values are calculated using GraphPad.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

For assays in 96 well plates 2500 cells are plated per well. After 24 hours, cells are treated with small molecule inhibitors and incubated for 72 hours at 37°C. Subsequently the cells are lysed and the ATP content is measured as an indicator of metabolically active cells using the CellTiter-Glo assay. IC₅₀ values are calculated using the GraphPad software. For assays in six well plates, 100,000 cells are plated per well. After 24 hours, cells are treated with small molecule inhibitors and incubated for varying time points. Cells are trypsinized and a suspension is made in 5 mL of phosphate buffered saline. 30 µL of this suspension is mixed with 30 µL of CellTiter-Glo reagent followed by a 10-minute incubation at room temperature. Luminescence is measured using EnVision 2104 Multilabel Reader and BioTek Synergy Neo Microplate Reader.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.