H3k36me3 9050

Wild-type ELF6 or mutant elf6-5 full genomic coding sequences were cloned into pEG104 vector^{35 (link)}. The demethylation assay was carried out as previously described^{16 (link)}. Briefly, N. benthamiana leaves were infiltrated with A. tumefaciens EHA105 strains carrying a functional wild-type 35S::YFP::ELF6 or mutant 35S::YFP::ELF6^A424V. Transfected nuclei were isolated after 48 h. Immunolabeling of fixated nuclei was performed using histone methylation-specific antibodies: H3K4me3 Millipore 07-473, 1:100; H3K9me2 Millipore 07-441, 1:200; H3K27me3 Millipore 07-449, 1:100; H3K27me2 Millipore 07-452, 1:200; H3K27me1 Millipore 07-448, 1:200; H3K36me3 Abcam 9050, 1:100. The modified histones were revealed by Alexa Fluor 555-conjugated goat anti-rabbit (Invitrogen, 1:200). Transfected cells were revealed by monitoring the YFP signal. After staining, the slides were mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratory) and then photographed with an OLYMPUS BX51 fluorescence microscope. Histone methylation levels were quantified by comparing staining density of a number of transfected 35S::YFP::ELF6 nuclei versus non-transfected nuclei in the same field. Image density was determined using ImageJ software. A negative result in our assay usually corresponds to 80% or less than total wild-type histone demethylase activity.