Idcr reagent

For total protein extracts, 100 mg of Arabidopsis leaves were homogenized in liquid nitrogen and 500 μl of SDS extraction buffer (4% w/v SDS, 100 mM Tris-HCl pH 7.6, 100 mM DTT, protease and phosphatase inhibitor cocktails) were added. Samples were boiled for 10 min, centrifuged at 12000 x g for 10 min, and the supernatant recovered. Nuclei were extracted from 2g fresh leaves using a previously described method [89 (link)]. Briefly, fresh leaves were chopped in 30 ml nuclear extraction buffer (2.0 M hexylene glycol (2-methyl-2,4-pentandiol), 20 mM PIPES/KOH pH7.0, 10 mM MgCl2 and 5 mM 2-mercaptoethanol, protease and phosphatase inhibitor cocktails), filtered through 5 layers of cheesecloth, and subjected onto a 30% / 80% percoll density gradient. After centrifugation at 2000 x g for 30 min, the layer between 30–80% percoll was collected, loaded on 30% percoll, and re-centrifuged at 2000 x g for 30 min. The pellet was collected, mixed with 100 μl SDS-extraction buffer, and boiled for 10 minutes. Samples were centrifuged (10000 x g, 10 min), and the supernatant was collected as the nuclear protein fraction. Protein concentrations were determined using Pierce 660 nm absorbance assay in presence of IDCR reagent following the manufacturer’s protocol (Thermo Fisher Scientific). Coomassie Brilliant Blue staining of membranes was used as loading control.