Synergy two multi mode microplate reader

The IC₅₀ value for the peptoid inhibitors were measured using the MTase-Glo methyltransferase assay kit (Promega). The peptoid was incubated in assay buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl₂, 0.1 mg/ml BSA, 1 mM DTT) and substrate (0–1000 μM). After a preincubation of 10 min, 200 nM PRMT1 or PRMT5 was added to the assay before adding in the substrate (AcH4-21) at 225 μM. After 15 min, the reaction was quenched with 0.5% TFA (0.1% final concentration), vortexed, and incubated for an additional 10 min. To detect product formation, the MTase-Glo reagent was added for 30 min at rt, followed by addition of the MTase-Glo detection solution for an additional 30 min. Finally, the luminescence signal was measured using a BioTek Synergy two multi-mode microplate reader. The values were converted to product concentration using a standard curve of SAH (0–5 μM). The IC₅₀ values were determined using Equation 1 and fit to a curve using GraFit 7.03. $\text{Fractional}\text{activity}\text{of}\text{PRMT}=1/\left(1+\left(\left[\text{I}\right]/{\text{IC}}_{50}\right)\right)$ IC₅₀ is the concentration of inhibitor resulting in 50% PRMT activity, and [I] is the inhibitor concentration.

For the Arabidopsis protoplast system, rosette leaves of 3-week-old Arabidopsis were used to prepare protoplasts. To-be-tested promoters were cloned into pGreenII 0800-LUC plasmid, while CsWRKY11 and CsNPR1 were cloned into pCHF3 plasmid. The reconstructed pGreenII 0800-LUC and pCHF3 plasmids were co-transferred into protoplasts via poly (ethylene glycol)-mediated transfection, and the transfected protoplasts were cultured at 22 °C overnight. Firefly and Renilla luciferase activities were then detected with the help of a dual-luciferase assay kit (Promega), and monitored with the Synergy two multi-mode microplate reader (Bio-Tek) according to the manufacturer’s instructions. For the living tobacco system, the reconstructed pGreenII 0800-LUC and pCHF3 plasmids were respectively transferred into Agrobacterium strain GV3101-pSoup-p19. After activation under light, the plasmid-transformed Agrobacterium cells were co-infiltrated into the 4-week-old tobacco leaves. After a 24-h cultivation under light, Agrobacterium cells-infiltrated tissues were frozen in liquid nitrogen, and dual-luciferase reporter assays were performed using the same method as described in the Arabidopsis protoplasts system.