Figure 6.

The afb4^D215N protein has reduced affinity for IAA7. Equivalent amounts of in vitro translated Myc-tagged TIR1, AFB4, or afb4^D215N proteins were incubated with GST-IAA7 protein attached to glutathione-agarose beads in the presence or absence of 50 µM IAA. After washing and elution from the agarose beads, the proteins were separated by polyacrylamide gel electrophoresis and blotted to nitrocellulose membranes. (A) The Myc-tagged receptor proteins were immunodetected using anti-c-Myc antibody and the GST-IAA7 input was visualized by Ponceau S staining. (B) The relative amounts of Myc-tagged proteins added. (C) The quantification of blot band density as presented in (A) by ImageJ. All the values were normalized to AFB4-Myc without IAA treatment. The experiment was repeated four times with similar results.