Fig. 2: Crosslinking with a functionalized CO5 shows high affinity LCO and short-chain CO binding to PhLYK9 and BdLYR2.

a Schematic representation of the cross-linkable biotinylated CO5 (CO5-biot). The triazine group forms a covalent bound with adjacent proteins and the biotin-labeled protein can be detected by Western-blotting (WB). b CO5-biot binding to 10 µg of N. benthamiana membrane proteins containing PhLYK9-YFP or PhLYK15-YFP. Membrane fractions were incubated with or without 10 nM CO5-biot and WB performed using sequentially anti-GFP antibodies and streptavidin on the same membrane. The arrowhead indicates the position of PhLYK9-YFP, whereas * indicates an N. benthamiana endogenously biotinylated protein. c, d CO5-biot binding to immunopurified PhLYK9, PhLYK15 or BdLYR2. 250 µg of membrane proteins containing the indicated proteins were incubated with or without 1 µM CO5-biot, before protein solubilization and purification using anti-GFP beads. Note that the endogenously biotinylated protein detected in membrane fractions (b) was not detected anymore after protein purification. Affinity of PhLYK9 (e) and BdLYR2 (f) for short-chain CO. Saturation experiments on 10 µg of membrane proteins using a range of concentrations of CO5-biot. Specificity of the PhLYK9 (g) and BdLYR2 (h) CO-binding sites for CO versus LCO. Ten µg of membrane proteins were incubated with or without 10 nM CO5-biot and a range of unlabeled LCO-V(C18:1,NMe,S). i, j CO5-biot binding to membrane proteins containing equivalent amounts of the indicated proteins. Membrane fractions were incubated with or without 10 nM CO5-biot. The blots represent data obtained four times in (e, f) and twice in (g, h).