AlphaScreen Assay for SIX1-EYA1 Interaction

The AlphaScreen assay was performed using GST-SIX1 and His-EYA1 proteins in the same small molecule pool as described previously.^{[19 (link)]} In brief, equal amounts (7.5 μL) of GST-SIX1 and His-EYA1 proteins were mixed with 5 μL glutathione donor beads and 5 μL nickel chelate acceptor beads (PerkinElmer, Waltham, MA, USA, #6760603M). After 30 min, equal volumes (2 μL) of individual compounds were added into the protein mixture and incubated at 16°C for 2 h. The AlphaScreen signals were collected by reading plates in an Envision Multilabel Reader (PerkinElmer, #2105-0010), and compounds that caused signal values to decrease significantly (<5000) were selected as candidates.