Fig. 4.
Auxin-enhanced SCFTIR1–Aux/IAA interaction may require modification of TIR1 or tightly associated proteins. (a) TIR1myc was immunoprecipitated (IP) from either mock- or auxin-treated tir1-1[TIR1myc] extract, washed, and eluted into tir1-1 extract. Pulldowns with domain II peptide were performed in these reconstituted extracts as indicated in the schematic. (b) As described for a but performed with additional controls for auxin carry through: in parallel with the immunoprecipitation stages, control beads (no antibody) were treated with and without auxin and combined with immunoprecipitated beads just before elution (–NAA IP with +NAA control, +NAA IP with –NAA control). (a and b) Lane 3 shows the full NAA-induced response where 10 μM NAA was added to the reconstituted extracts before pulldown. Lanes 4–6 in show the levels of TIR1myc in reconstituted extracts made with tir1-1 extract and –NAA IP, +NAA IP, and no immunoprecipitation, respectively. (c) Mean densitometric measurements of mock- and NAA-pretreated TIR1myc in pulled-down with domain II peptide as a percentage of the full NAA-induced response (n = 4). n, the number of independent replicates; *, the position of the TIR1myc band. (Error bars, SEM.)