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. 2015 Nov 6;27(11):3128–3142. doi: 10.1105/tpc.15.00605

Figure 5.

Figure 5.

GFP-SMXL6, 7, and 8 Fusion Proteins Are Polyubiquitinated and Degraded in Response to rac-GR24 Treatment in Arabidopsis.

(A) Ubiquitination of GFP-SMXL6, GFP-SMXL7, and GFP-SMXL8 in protoplasts. Arabidopsis (Col-0) protoplasts were transformed with plasmids encoding each fusion protein, incubated for a 12-h period of protein synthesis, then pretreated with 50 µM MG132 for 1 h and treated with 40 µM rac-GR24 or 40 µM KAR1 for 1 h. Proteins were isolated for immunoprecipitation with agarose-conjugated anti-GFP monoclonal antibody followed by immunoblotting with antiubiquitin antibody (upper panel) or anti-GFP (lower panel).

(B) Levels of GFP-SMXL6, GFP-SMXL7, and GFP-SMXL8 proteins in wild-type protoplasts after plasmid transformation and treatment with 40 µM rac-GR24 or 40 µM KAR1 for the times indicated. Proteins were detected by immunoblotting with anti-GFP monoclonal antibody. Relative amounts of proteins were determined by densitometry and normalized to loadings determined by Ponceau staining (red) and expressed relative to the value at zero time.

(C) Ubiquitination of SMXL6-GFP and SMXL6D-GFP in wild-type (Col-0) Arabidopsis containing 35S:SMXL6-GFP and 35S:SMXL6D-GFP transgenes. Seedlings were treated after 10 d of growth with 50 µM MG132 for 1 h, then with 2 µM rac-GR24 in 0.5× MS liquid medium for 10 min. Proteins were detected as in (A).

(D) Levels of SMXL6-GFP and SMXL6D-GFP proteins in wild-type (Col-0) Arabidopsis containing 35S:SMXL6-GFP and 35S:SMXL6D-GFP transgenes. Seedlings were treated after 10 d of growth with 2 µM rac-GR24 in 0.5× MS liquid medium for the times indicated. Proteins were detected as in (B).

(E) Ubiquitination of SMXL6-GFP in wild-type (Col-0), max2-1, and d14-1 transgenic plants each expressing 35S:SMXL6-GFP. Seedlings were treated after 10 d of growth with 50 µM MG132 for 1 h, then with 2 µM rac-GR24 in 0.5× MS liquid medium for 10 min. Proteins were detected as in (A).

(F) Level of SMXL6-GFP in wild-type (Col-0), max2-1, and d14-1 transgenic plants each expressing 35S:SMXL6-GFP. Seedlings were treated after 10 d of growth with 2 µM rac-GR24 in 0.5× MS liquid medium for the times indicated. Proteins were detected as in (B).