A formaldehyde dehydrogenase (FDH)-coupled
demethylase assay was used to determine demethylase activity and to
select potent inhibitors. All inhibitors were dissolved in dimethyl
sulfoxide (DMSO) at various concentrations and added to the mixture
such that the final DMSO concentration was 5%. The reagents for the
demethylase reactions were dissolved in HEPES buffer (50 mM, pH 7.5),
with the exception of Fe(II) solutions, which were made using (NH4)2Fe(SO4)2 dissolved in 20 mM HCl to make
a 400 mM stock solution. All reagents were stored at −30 °C.
FDH, NAD+, TKQTARK(Me)3STGGKAPR (H33–17K9me3), STGGVK(Me)3KPHRY (H331–41K36me3),
or ARTK(Me)3QTARK(Me)2STGGKAPRKQLATKA
(H31–24K4me3K9me2) peptides (Kelowna Int. Sci. Inc.),
DMSO, and the demethylase enzyme were added first to 96-well black
immuno plate (SPL Life Science) and incubated together on ice for
15 min. Then, the plate was put into a FLUOStar OPTIMA ELISA reader
(BMG LABTECH) at 37 °C, and the reaction was started by adding
ascorbic acid (ascorbate), Fe(II), and AKG to final concentrations
of 50 mM HEPES, pH 7.5, 2 μM of KDM4B, 5% DMSO, 0.01 U FDH (Sigma),
1 mM NAD+, 1 mM AKG, 2 mM ascorbate, 50 μM Fe(II), and various
concentration of H3K9me3 peptide; the final volume was 50 μL.
Each reaction was incubated at 37 °C for 30 min, and the production
of NADH was detected by fluorescence (ex 360/em 470).
demethylase assay was used to determine demethylase activity and to
select potent inhibitors. All inhibitors were dissolved in dimethyl
sulfoxide (DMSO) at various concentrations and added to the mixture
such that the final DMSO concentration was 5%. The reagents for the
demethylase reactions were dissolved in HEPES buffer (50 mM, pH 7.5),
with the exception of Fe(II) solutions, which were made using (NH4)2Fe(SO4)2 dissolved in 20 mM HCl to make
a 400 mM stock solution. All reagents were stored at −30 °C.
FDH, NAD+, TKQTARK(Me)3STGGKAPR (H33–17K9me3), STGGVK(Me)3KPHRY (H331–41K36me3),
or ARTK(Me)3QTARK(Me)2STGGKAPRKQLATKA
(H31–24K4me3K9me2) peptides (Kelowna Int. Sci. Inc.),
DMSO, and the demethylase enzyme were added first to 96-well black
immuno plate (SPL Life Science) and incubated together on ice for
15 min. Then, the plate was put into a FLUOStar OPTIMA ELISA reader
(BMG LABTECH) at 37 °C, and the reaction was started by adding
ascorbic acid (ascorbate), Fe(II), and AKG to final concentrations
of 50 mM HEPES, pH 7.5, 2 μM of KDM4B, 5% DMSO, 0.01 U FDH (Sigma),
1 mM NAD+, 1 mM AKG, 2 mM ascorbate, 50 μM Fe(II), and various
concentration of H3K9me3 peptide; the final volume was 50 μL.
Each reaction was incubated at 37 °C for 30 min, and the production
of NADH was detected by fluorescence (ex 360/em 470).