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. 2010 Jan 26;285(15):11557–11571. doi: 10.1074/jbc.M109.027011

FIGURE 8.

FIGURE 8.

Epitope mapping for mAbs and interaction of AtGID1 receptors from A. thaliana QUAD with recombinant DELLA proteins using antibody sandwich immunoassays. AtGAIn, AtRGL1n, and AtRGL2n were incubated with either extraction buffer (solid symbols) or QUAD extract containing AtGID1 receptors (open symbols) for 1 h. DELLA proteins or their complexes with AtGID1 receptors were captured by specific mAb AF2, AB8, and BB7 for AtGAIn, AtRGL1n, and AtRGL2n, respectively. All assays were carried out for AtGAIn (□), AtRGL1n (▵), and AtRGL2n (○) mixed with QUAD extract and AtGAIn (■), AtRGL1n (▴), and AtRGL2n (●) mixed with extraction buffer. A, the epitopes of mAb BC9 (reactive to the DELLA motif of all DELLAs) and AD7 (reactive to the VHYNP motif of AtRGL1n, AtRGL2n, and AtRGL3n) were identified by synthesizing the respective peptides with replacement of each individual amino acid with alanine. The amino acids marked with asterisks are involved in interactions with AtGID1a. B, the AtGID1 receptors in QUAD extracts bind to recombinant DELLA proteins. The AtGID1 in resultant complexes was detected with rabbit polyclonal anti-AtGID1c antibody and peroxidase-labeled goat anti-rabbit secondary antibody. C, the interaction of AtGID1 receptors with DELLA proteins inhibits the binding of biotin-labeled mAb BC9, which recognizes the DELLA motif of all DELLAs examined. mAb BC9 was detected by peroxidase-labeled streptavidin. D, the interaction of AtGID1 receptors with AtRGL1n and AtRGL2n reduces the binding of biotin-labeled mAb AD7, which recognizes the VHYNP motif of AtRGL1n and AtRGL2n. mAb AD7 does not recognize the VHYNP motif of AtGAIn except after binding of AtGID1 receptors to AtGAIn. mAb AD7 was detected by peroxidase-labeled streptavidin. The native Western blotting confirmed the ELISA result (inset in the left panel); AtGAIn incubated with extraction buffer (lanes 1 and 2), AtGAIn incubated with QUAD extract (lane 3), and QUAD extract control (lane 4) were separated on a 7.5% native polyacrylamide gel and then transferred to polyvinylidene difluoride membrane. An AtGAIn (band marked with asterisk in lane 1) was detected with AtGAIn-specific mAb AF2; the AtGID1-AtGAIn complex (band marked with # in lane 3) was detected with mAb AD7; and mAb AD7 did not detect AtGAIn incubated with extraction buffer (lane 2) or any corresponding components in QUAD extract control (lane 4).