Table 2.

Ultrahigh-Throughput Screening Protocol

Step	Parameter	Value	Description
1	Dispense controls	5 μL/well	Cell lysate (22.9 μg/mL) expressing VF-NSD3S mixed with TR-FRET antibodies (anti-Flag-Tb and anti-GST-d2)
2	Dispense reaction buffer	5 μL/well	A mixture of cell lysate (22.9 μg/mL) coexpressing VF-NSD3S and GST-MYC with TR-FRET antibodies (anti-Flag-Tb and anti-GST-d2)
3	Library compounds	100 nL/well	19.6 μM final from 1 mM stock in DMSO
4	Incubation time	16 h	Plates were incubated at 4°C for 16 h
5	Assay readout	Excitation 337 nm (laser), Emission 1 615 nm, Emission 2 665 nm; dual mirror D400/D630	Time-resolved fluorescence; Envision Multilabel plate reader

Step Notes

1. 1,536-well black solid bottom plates (Corning Costar, #3724) were used. FRET buffer (20 mM Tris-HCl, pH 7.0, 50 mM NaCl, and 0.01% nonidet P-40) was used to mix with assay components. One column (32 wells) was used for controls. MultiDrop Combi dispenser (Thermo Scientific) was used for dispensing.

2. Reaction buffer was prepared in parallel with controls.

3. 0.1 μL compounds (1 mM stock in DMSO in 384-well plates) were added using a Pintool (VP Scientific) integrated with a Beckman NX (Beckman Coulter, Brea, CA).

4. Plates were incubated at 4°C for 16 h with top plates lidded.

5. Plates were read using EnVision Multilabel plate reader with 50 μs delay in the TR-FRET signal measurement.

 DMSO, dimethyl sulfoxide.