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. 2015 Mar 20;27(3):806–822. doi: 10.1105/tpc.114.135210

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© 2015 American Society of Plant Biologists. All rights reserved.

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Figure 2. — Subcellular Localization of ROP10:GFP and Mutant Forms in N. benthamiana Leaf Epidermal Cells.

(A) ROP10:GFP, ROP10CA:GFP, and ROP10DN:GFP were localized exclusively at the PM, whereas free GFP was distributed in the PM, cytoplasm, and nuclei. Bars = 50 μm.

(B) Inhibition of S-acylation of ROP10:GFP, ROP10CA:GFP, and ROP10DN:GFP by a treatment with 2-bromopalmitate resulted in release of the expressed proteins from the PM. Fluorescence is observed in the cytoplasm and nuclei (arrowheads). Bars = 50 μm.

(C) ROP10 proteins with mutations in the GC-CG box (ROP10^C203S:GFP, ROP10^C197S:GFP, and ROP10^C197+203S:GFP) and ROP10CA mutant with mutations (ROP10CA^C197+203S:GFP) showed reduced association with the PM, and fluorescence is observed in the cytoplasm and nuclei (arrowheads). Bars = 50 μm.

(D) Immunoblotting using GFP monoclonal antibody. Soluble (S) and insoluble membrane (P) fractions were separated from N. benthamiana leaf cells overexpressing different forms of ROP10 proteins. ROP10, ROP10CA, and ROP10DN were detected in the membrane fraction, and free GFP was detected in both the soluble and membrane fractions.