Anti flag hrp conjugated antibody

ELISAs for binding affinity and specificity were performed as described previously^{17 (link)} with minor modifications. Briefly, biotinylated oligonucleotides were bound to Pierce streptavidin-coated high capacity plates (ThermoFisher) followed by blocking with 3% BSA and incubation with full-length recombinant human GST-tagged UHRF1 (Abnova, catalogue no. H00029128-P01) and DDX24 (Abnova, catalogue no. H00057062-P01), HIS-tagged SMARCA4 (Abcam, catalogue no. ab82237), RBM22 (OriGene, TP760056) and Myc/DDK-tagged DDX1 (OriGene, TP308769) in ELISA buffer (100 mM KCl and 50 mM KH₂PO₄, pH 7.4). After three washes with the ELISA buffer, detection was achieved with an anti-GST HRP (horseradish peroxidase)-conjugated antibody (Abcam, catalogue no. ab3416) diluted to 1:5,000, anti-FLAG HRP-conjugated antibody (Abcam, ab1238,) diluted to 1:15,000 or anti-HIS HRP-conjugated antibody (BioLegend, catalogue no. 652503) diluted to 1:3,000 in an ELISA buffer that contained 3% BSA and 3,3′,5,5′-tetramethylbenzidine ELISA substrate (slow kinetic rate) (Abcam, ab171525). Signal intensity was measured at 450 nm on a SPECTROstar nano microplate reader (BMG Labtech). K_d values were calculated from binding curves assuming a one-site binding model in GraphPad Prism, and standard error of means from three replicates are reported.

Full-length NAM-A1 alleles were cloned into the Gateway binary vector pGWB12, with an N-terminal FLAG tag [60 (link)]. These were transformed into Agrobacterium (strain LBA4404) and co-infiltrated into 3-week-old N. benthamiana leaves alongside the silencing inhibitor P19 at a total OD of 0.4, and individual OD of 0.2 per construct. Leaves were harvested 3 days post-infiltration and snap-frozen in liquid N₂. Leaf tissue was ground in liquid N₂, protein was extracted as described above, and run on a 12% SDS-PAGE gel. The protein was transferred to a nitrocellulose membrane and probed with an anti-FLAG HRP-conjugated antibody (Abcam). Total protein input was visualised using a Ponceau stain.
The NAM-A1 alleles were also patch-infiltrated into N. benthamiana leaves at the OD detailed above. The infiltrated patches were scored for cell death at 5 days post-infiltration, as detailed in Maqbool et al. 2015 [34 (link)]. Briefly, images were taken of the abaxial and adaxial sides of the leaves using U.V. and white light, respectively. The infiltrated patches were then scored on a discrete scale from 0 to 6 based on the intensity of the fluorescence or chlorosis. Infiltrations were repeated on at least 20 independent leaves per allele in a randomised pattern around the leaf.

Protein extracts were prepared as described above and incubated for 5 min. at 100 °C. Different amounts of proteins were loaded on 4–20% gradient acrylamide gel and transferred onto PVDF membrane. After incubation with corresponding antibodies, the protein analysis was performed using ECL-PLUS kit (GE-Healtcare) as described before and VersaDoc apparatus (Bio-Rad) as previously described^{35 (link)}. Primary antibodies used: Anti-FLAG -HRP conjugated antibody (abcam; 1:10000), Anti Actin (abcam 1:500) and Anti RAD-51 (1:1000).