Fig 1: Detection of H2O2 in HRP assay. A mixture of CeLLestialTM Red, HRP, 9a (25 µM), and LSD1 (0.5 µg/well) were deposited in all wells. The assay plate was incubated at 25°C for 3 h. Absorption was measured with ARVO X5 (filters; Ex. = 531/25 nm, Em. 595/60 nm) every 3 min for 3 h. Experiments were run in triplicate.
Fig 2: Enzymatic reaction of 9a with active LSD1 or heat-denatured LSD1 (90°C for 5 min). Enzymatic reactions were performed in LSD1/HRP buffer, containing 25 µM 9a, 5 ng/µl LSD1 (active or denatured). Absorption was measured with ARVO X5 (filters; 405/10 nm) every 5 min for 3 h at 25°C. The results are shown as mean ± SD. (n = 3). (A) Time-dependent curve (B) Absorption of 9a after enzymatic reaction for 180 min. *p<0.0001 (Student’s t test).
Fig 3: Enzymatic reaction of 9a with LSD1 in the presence or absence of LSD1 inhibitor. Enzymatic reactions were performed in LSD1/HRP buffer, containing 25 µM 9a, 5 ng/µl LSD1 after pre-incubation in the presence or absence of 160 nM GSK-LSD1 for 30 min. Absorption was measured with ARVO X5 (filters; 405/10 nm) after 3 h incubation at 25°C. The results are shown as mean ± SD (n = 3). *p<0.001, **p<0.0001 (Bonferroni-type multiple t test).
Fig 4: Synthesis of various LSD1 artificial substrates.
Fig 5: (A) FAD-dependent demethylation mechanism of methylated lysine by LSD1. (B) Our strategy to detect LSD1 activity through β-elimination.
Supplier Page from Enzo Life Sciences, Inc. for LSD1 (human), (recombinant)