| Assay Method Information | |
| | USP9X Enzyme Assay |
| Description: | USP9X enzyme assays were set up in a 96 well plate to assess the abilities of Compound 3A and Compound 4A to inhibit the activity of USP9X, respectively. Ubiquitin-rhodamine110 was used as the substrate and ubiquitin aldehyde was included in the assay as a positive control. Ubiquitin aldehyde binds covalently to the thiol group of the active site Cys of USP9X. The activity of USP9X was completely abolished by incubation with ubiquitin aldehyde. The activity of USP9X was reduced approximately 60% with the higher concentrations of Compound 2A, Compound 3A, and Compound 4A (>10 μM). The partial inhibition of USP9X was not due to the lack of solubility of these compounds at high concentrations but instead was shown to have a mechanistic basis. |
| Affinity data for this assay | |
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