Novex prestained ladder

The EpiQuik Total Histone Extraction Kit (Epigentek) was used to extract histones from honeybee ovary tissue. Qubit fluorometer and protein assay kits (Invitrogen) were used to estimate protein concentration. Ten micrograms of protein were separated on a 12% SDS PAGE gel at 200 V for 1 h in Tris-Glycine-SDS electrophoresis buffer and run alongside a molecular weight marker (Novex prestained ladder [Invitrogen]). Proteins were transferred to nitrocellulose membrane in Towbin’s buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3, 20% methanol), the membrane was blocked in 2% bovine serum albumin in TBS-T before incubation with H3K27me3 antibody (Abcam 6002) (1:200) at 4 °C overnight. The membrane was washed and incubated with HRP-conjugate anti-mouse (Jackson Immunochemicals) (1:1,000) at room temperature for 1 h. The chemiluminescent reaction step was performed using the Pierce ECL Western Blotting substrate (Thermofisher). The blot was imaged using the Fuji LAS-3000 ECL imaging system. After detection of H3K27me3, the membrane was stripped for 10 min in stripping buffer (200 mM Glycine, 0.01% SDS, 0.1% Tween) and washed before incubation with the anti-Histone H3 Polyclonal Rabbit (abcam 1791) (1:1,000) and a second chemiluminescent reaction step.

Non-infected, wild type (WT) TMV and MBP TMV infected N. benthamiana leaves were harvested for western blotting 16 days after inoculation. Approximately 30 mg of leaf material was ground in liquid nitrogen, combined with 100 μl of 2X Laemmli loading buffer and then boiled for 5 min to denature the proteins. After centrifugation at 16,000 rpm, 20 μl was loaded onto 15% SDS-PAGE gels, along with 15 μl of Novex prestained ladder (Invitrogen, Paisley, UK). Gels were electroblotted onto Immobilon-P membrane (Millipore, Watford, UK). The Immobilon-P membrane was blocked by incubating in 1X PBS, 1% BSA and 0.05% Tween, for 1 h. Anti-TMV antibodies raised in rabbits were added to the membranes in blocking solution at a 1/10000 dilution and incubated with shaking at room temperature for 1 h. After washing, an anti-rabbit IgG alkaline phosphatase conjugate was added to the blots at a concentration of 1/1000 in blocking buffer (A8025-1ML; Sigma, Dorset, UK). After incubation at room temperature under shaking conditions the blots were washed and covered with BCIP/NBT (B1911; Sigma, Dorset, UK). The blots were left to develop for 10 min, after which banding was visible. The developed blots were scanned and saved as jpeg images.