ibr5 does not accumulate AXR3NT-GUS. (A) 8-day-old Col-0 (Wt), ibr5-1, tir1-1, tir1-1 ibr5-1, and axr1-3 seedlings carrying HS:AXR3NT-GUS  were heat shocked for 2 hours, treated with mock (ethanol) or 100 nM IAA for the indicated time, then stained for GUS activity. (B) Auxin-response defects of HS:AXR3NT-GUS lines. Lengths of primary roots of 8-day-old seedlings grown under yellow-filtered light at 22°C on medium supplemented with various concentrations of 2,4-D are shown. Error bars represent standard errors of the means (n = 20). (C) 8-day-old Col-0 (Wt) and ibr5-1 carrying HS:axr3-1NT-GUS  were heat-shocked for 2 hours, mock (ethanol) treated or treated with 10 μM IBA for 40 minutes, then stained for GUS activity. (D) 8-day-old Col-0 (Wt), ibr5-1, tir1-1, and tir1-1 ibr5-1 carrying HS:AXR3NT-GUS  were heat shocked for 2 hours. Midway through a 2-hour heat shock, DMSO (mock) or 50 μM MG132 treatment was initiated. Seedlings were stained for GUS activity 2 hours after return to room temperature. Separate experiments revealed that inclusion of DMSO during the heat shock (included as an MG132 carrier in panel D) resulted in more intense AXR3NT-GUS staining (L.C.S., unpublished), which could account for the higher apparent GUS activity in panel D when compared to panel A.