E3 Ligase Binding Assay Protocol

The E3 ligase in vitro binding assays were adapted from Nowak et al. (2018) (link). Briefly, a day before compound treatment, cells stably expressing BRD4_BD2-GFP with mCherry reporter were seeded at 30–50% confluency in 384-well plates (3764, Corning) with 50 μL FluoroBrite DMEM media (Gibco, A18967) containing 10% FBS per well. Compounds and either 250 nM AT-1 (Gadd et al., 2017 (link)) for the VHL engagement assay or 100 nM dBET6 (Winter et al., 2017 (link)) for CRBN engagement assay, were dispensed using D300e Digital Dispenser (HP) normalized to 0.5% DMSO and incubated with cells for 5 h. The assay plate was imaged immediately using Acumen eX3/HCl (TTPLabtech) High Content Imager with 488 and 561 nm lasers in 2 × 1 μm grid per well format. The GFP/RFP ratio of the resulting images was analyzed using a CellProfiler pipeline (Carpenter et al., 2006 (link)). The GFP/mCherry ratio was normalized to DMSO and fitted in GraphPad Prism 7 using variable slope equation.