Enspire 2300 multimode reader

To study the interaction of MLL1 with the RBBP5/ASH2L or WDR5/RBBP5/ASH2L complexes with AlphaScreen assays, 0.5 μm His‐tagged RBBP5 protein, 0.5 μm His‐tagged ASH2L protein and (if needed) 0.5 μm His‐tagged WDR5 were pre‐incubated for 30 min at 4 °C to form the corresponding complexes. A 10 μL aliquot of the complexes was loaded in each well of a microplate (1/2 Area plate™‐96; PerkinElmer). Then, 10 μL of 0.5 μm GST‐tagged MLL1‐SET was added and incubated for 1 h at 22 °C. Afterward, 0.8 μg nickel‐chelate acceptor beads (PerkinElmer) and 0.8 μg glutathione donor beads (PerkinElmer) were added and incubated for another 1 h in the dark at 22 °C. As negative controls, empty beads or beads incubated with 0.5 μm GST protein were included. The AlphaScreen light signal was measured with an EnSpire™ 2300 Multimode reader (PerkinElmer). The experiments were conducted in AlphaLISA Universal Buffer (PerkinElmer AL001C) containing PBS (10 mm phosphate, 137 mm NaCl, 2.7 mm KCl) pH 7.2, 0.1% BSA, and 0.01% Proclin‐300.

All the chemicals used in this study were of analytical grade. Organic solvents, including 1-butanol, acetone, chloroform (also for NMR), and ethyl acetate, were purchased from KANTO CHEMICAL CO., INC., Japan. Chemicals, such as hydrochloric acid, K₂HPO₄, KH₂PO₄, L-tyrosine, silica gel, octadecyl silica gel, and tris (hydroxymethyl) aminomethane, were bought from NACALAI TESQUE, INC., Japan. Standard substances, including β-arbutin, caffeic acid, and ursolic acid, and tricine were obtained from Tokyo Chemical Industry, Japan. Enzymes, such as collagenase, elastase, and tyrosinase, including N-succinyl-Ala-Ala-Ala-p-nitroanilide (SANA) and dimethyl sulfoxide (DMSO), were acquired from Sigma-Aldrich, Germany. DMSO and methanol (NMR grade) were purchased from ISOTEC^®, Sigma-Aldrich, Germany. A peptide: MOCAc-PRO-Leu-Gly-Leu-A₂pr(Dnp)-Ala-Arg-NH₂, was ordered from Peptide Institute, Inc., Japan. Instruments used included HPLC (SCL-10A sp/RID-6A/c-R3A; SHIMADZU, Kyoto, Japan), Fluorescence microplate reader (Enspire 2300 Multimode reader; PerkinElmer, Waltham, MA, USA), HR-ESI-MS (LTQ orbitrap XL; Thermo Fisher Scientific, Waltham, MA, USA), Infrared Spectrophotometer (FT-720, HORIBA), Nuclear Magnetic Resonance Spectrometer (ECA-500/600, JEOL), and UV-Vis microplate reader (Multiskan Go; Thermo Scientific, Waltham, MA, USA).

A CCK‐8 assay (Dojindo) was used to determine the cell viability following the manufacturer’s instructions. RAW264.7 cells were treated with various concentrations of (R)‐salbutamol for one hour prior to LPS induction (100 ng/mL). Cells were then incubated for a further 2 hours with the addition of 10 μL of CCK‐8. Cells were visualized at 450 nm with an Enspire‐2300 Multimode Reader (PerkinElmer).