Figure 7.

HIS_6x-HA_3x-IAA1 16KR protein levels are regulated by auxin and the UPS in vivo. A, 16KR and wild-type (Wt) IAA1-expressing transgenic seedlings were treated with 5 μm IAA or solvent or 50 μm MG132 and corresponding solvent for 6 h. HIS_6x-HA_3x-IAA1 proteins were visualized by anti-HA immunoblotting, with Ponceau S-stained membranes shown as loading controls; 80 μg of total protein is loaded for each sample. B, In vivo ubiquitination of wild-type and 16KR proteins was examined by DPDs from plant extracts of MG132-treated seedlings. HIS_6x-HA_3x-IAA1 was immunoprecipitated from approximately 5 mg of total protein using anti-HA beads. Col-0 extract, a nontransgenic line, was used as a negative control. Protein was eluted from anti-HA beads in denaturing urea buffer, recaptured on Co²⁺ beads, and finally eluted in 1× Laemmli sample buffer; 20% and 80% of the eluted protein were loaded for anti-HA and anti-UBQ, respectively, and 80 μg of total protein was loaded for input for both blots. Unmodified HIS_6x-HA_3x-IAA1 is indicated with arrows on the right, and molecular mass markers in kilodaltons are labeled on the left. Two exposures for the anti-HA blot are shown to show equal loading of the unmodified forms (short exposure) and visualize the slower migrating species (long exposure).