FIGURE 6.
Post-translational regulation of SCFTIR1 accumulation in wild-type plants. Ler np::TIR1-HAStrep seedlings were grown in liquid MS for 7 days. A, seedlings were incubated for 6 h with the proteasome inhibitor MG-132 (50 μm (+) or with DMSO (-)). Total protein extracts were analyzed on immunoblots with anti-HA, anti-ASK, anti-CUL1, and anti-HSC70 antibodies. Immunodetection of cytosolic/nuclear HSC70 is used to evaluate relative loading of the samples. The open and filled arrowheads correspond to unmodified and RUB-modified CUL1, respectively. B, densitometric analysis of TIR1-HAStrep, ASK1, and total CUL1 levels (unmodified plus RUB-modified) detected by immunoblots of samples with and without MG-132 treatment was performed on three independent experiments. Error bars indicate S.D. Differences between untreated control and MG-132-treated samples were evaluated for statistical significance using Student's t test; *, p < 0.001. C, TIR1-HAStrep was affinity-purified on StrepTactin-Sepharose 6 h after application of auxin (5 μm 2,4-D) and/or MG-132 (50 μm). Protein extracts were analyzed on immunoblots using the anti-HA and anti-ubiquitin antibodies. The filled dot indicates unmodified TIR1-HAStrep, and the open arrowheads indicate modified TIR1-HAStrep.