Fig. 1: In vitro interaction analysis of GID1 and DELLA proteins from grapes. | Communications Biology

Fig. 1: In vitro interaction analysis of GID1 and DELLA proteins from grapes.

From: Identification of a new gibberellin receptor agonist, diphegaractin, by a cell-free chemical screening system

Fig. 1

a Structures of GAs. b Principle of interaction analysis between GID1 and DELLA using the AlphaScreen system. Biotinylated GID1 protein binds to streptavidin on donor beads with an extremely specific and high affinity. The protein A-coated acceptor beads were combined with FLAG-tagged DELLA protein using an anti-FLAG antibody. The GID1-DELLA complex forms a large complex with two types of beads through the antibody and streptavidin. After illumination at 680 nm, the donor bead converts ambient oxygen to singlet oxygen, and the singlet oxygen is transferred across to activate the acceptor bead and subsequently emits light at 520–620 nm. c Synthesis of VvGID and VvDELLA proteins. Biotinylated VvGID1 and FLAG-tagged VvDELLA proteins were synthesised using a wheat cell-free system. The synthesis of these proteins was confirmed by immunoblotting using anti-biotin and anti-FLAG antibodies. d Interaction assay between VvGID1a and VvDELLA proteins. The interaction of VvGID1a with VvDELLA proteins was analysed using the AlphaScreen system using GAs at various concentrations. Data are shown as mean and individual data points from three independent experiments. e Interaction assay between VvGID1b and VvDELLA proteins. The interaction of VvGID1b with VvDELLA proteins was analysed using the AlphaScreen system using GAs at various concentrations. Data are shown as mean and individual data points from three independent experiments.

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