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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Nat Chem Biol. 2021 Nov 15;18(1):56–63. doi: 10.1038/s41589-021-00898-0

Extended Data Fig. 5 ∣. NSD2-PWWP1 DNA binding and displacement assay.

Extended Data Fig. 5 ∣

A fluorescence polarization (FP)-based DNA displacement assay was used to test the ability of (a) UNC6934 and (b) UNC7145 to displace dsDNA from NSD2-PWWP1. Maximum FP signal was generated using 500 nM of NSD2-PWWP1 and 5 nM fluorescein-labelled dsDNA (32 bp) as described in materials and methods and was considered as 100%. Various concentrations of each compound up to 25 μM was tested for displacement of the dsDNA. UNC6934 and UNC7145 showed no significant effect on dsDNA binding to NSD2-PWWP1. In a complementary experiment, (c) binding of dsDNA to NSD2-PWWP1 in the presence of 20 μM (●) UNC6934, and 20 μM (■) UNC7145 or (▲) no compound was tested at various concentrations of NSD2-PWWP1 up to 2.5 μM. No significant difference in the pattern of dsDNA binding to NSD2-PWWP1 was observed in the presence of either compound, indicating UNC6934 and UNC7145 are not binding to the dsDNA binding site. In addition, (d) the unlabeled dsDNA (32 bp) was tested as a control for displacing the fluorescein-labelled dsDNA. Unlabeled dsDNA was able to compete with the labelled dsDNA for the dsDNA binding site with a displacement constant (Kdisp) of 300nM. The final DMSO concentration was 0.5% in all experiments. Experiments were performed in triplicate (n = 3).