Pierce glutathione magnetic agarose beads

For pulldown experiments, 10 µl of glutathione magnetic agarose beads (Pierce Glutathione Magnetic Agarose Beads, Thermo Scientific) were equilibrated by washing them two times with wash buffer (100 mM Sodium Phosphate pH 7.2, 300 mM NaCl, 1 mM DTT, 0.01% (v/v) IGEPAL). Normalized E. coli clarified lysates or purified proteins were mixed, according to the experiment, added to the washed beads and incubated on an end-over-end rotator for 1 hr at 4°C. Beads were washed five times in 1 ml wash buffer. Bound proteins were eluted by adding 100 µl Laemmli buffer. Samples were analysed by western blotting or Coomassie staining.

DAP-seq was performed as an outservice by Bluescape Scientific, Hebei, China. Genomic DNA (gDNA) was extracted from leaves, and a DAP library was constructed after fragmentation of the gDNA. Recombinant VvMADS28 protein was obtained by engineering the VvMADS28 cDNA into the pGEX 4 T expression vector, followed by expression and purification following the manufacturer’s specifications (Pierce™ Glutathione Magnetic Agarose Beads, Thermo Fisher). VvMADS28 protein and the gDNA library were incubated in vitro and DNA bound to VvMADS28 was isolated as described elsewhere [53 (link), 54 (link)]. DNA obtained after affinity purification and elution was subjected to paired-end sequencing on an Illumina HiSeq platform. Quality-filtered reads were aligned to a Grapevine genome sequence (https://urgi.versailles.inra.fr/Species/Vitis/Annotations) by Bowtie2 [55 (link)]. Conserved motifs within peak regions were identified using MEME [56 (link)].

For the enrichment of ubiquitin chains, GST-TRABID NZF 1–3 was used while GST-TRABID NZF 1-3^TY>LV (ubiquitin binding deficient mutant) and/or GST were used as control baits. All recombinant proteins were expressed in E. Coli BL21(DE3)RIL and purified a previously described^{54 (link)}. For each pull down condition, 10 μg of GST tagged protein was bound to Pierce Glutathione Magnetic Agarose Beads (Thermo Fisher, 78,602), for 1 h at RT. Cells in 10 cm³ dishes were lysed in 500 μl of GST-TRABID NZF 1–3 IP Lysis Buffer (150 mM NaCl, 50 mM Tris pH 7.4, 5 mM DTT, 2 mM NEM, 10 mM iodoacetamide, 1% v/v Triton X-100) for 20 min before being clarified by centrifugation (13000 rpm for 15 min at 4 °C). 60 μl of lysate was collected for the input sample. For each pull down condition, 110 μl of lysate was added to 50 μl of conjugated beads in 500 μl of Pull-Down Buffer (150 mM NaCl, 50 mM Tris pH 7.4, 5 mM DTT, 2 mM NEM, 10 mM iodoacetamide, 100 μM ZnCl2, 0.1% (v/v) Nonidet P-40, 0.5 mg/ml BSA) overnight at 4 °C . Beads were then washed four times in Wash Buffer (250 mM NaCl, 50 mM Tris pH 7.4, 5 mM DTT, 0.1% (v/v) Nonidet P-40) for 5 min, before addition of 2X LDS sample buffer supplemented with 100 mM DTT.