LanthaScreenTM Eu kinase binding assay for ALK5

As a verified method to detect kinase inhibitor, LanthaScreen^TM Eu kinase binding assay was used to analyze the interaction of the selected flavonoids with ALK5⁴⁷. Kinase buffer consisting of 50 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (pH 7.5), 10 mM MgCl₂, 1 mM ethyleneglycoltetraacetic acid, and 0.01% Brij-35 was used, in which 1% DMSO was added to form compound dilution buffer. A mixture of fourfold serially diluted compounds (5 μl), kinase/antibody solution (5 μl of GST-ALK5 (15 nM) and Eu-anti-GST antibody (6 nM); Invitrogen, Carlsbad, CA, USA) and kinase tracer 178 (5 μl of 30 nM; Invitrogen, Carlsbad, CA, USA) was added to each assay well in a low-volume 384-well plate (Corning Part #3676) for 1-h incubation at room temperature. Fluorescence resonance energy transfer signal was read using a Tecan Infinite F-500 plate reader. The emission ratio was defined as the acceptor/tracer emission (665 nm) divided by the antibody/donor emission (615 nm). IC₅₀ value was determined based on the sigmoidal dose−response curve that was generated using GraphPad Prism software.