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. Author manuscript; available in PMC: 2015 Feb 19.
Published in final edited form as: Nat Commun. 2014 Aug 19;5:4687. doi: 10.1038/ncomms5687

Figure 2. Mutating serine 75 and 77 to alanine promotes SUMOylation and increases CES protein stability.

Figure 2

(a) In vitro kinase assays with affinity purified recombinant CESwt-GST or CESS75A+S77A-GST protein, [γ-32P]ATP and total plant extracts from arabidopsis flowers. (b) Confocal microscopic images showing CESS75A+S77A-YFP localization in different tissues of two-week-old plants. Scale bars: hypocotyls: filter = 40 μm, magnified = 5 μm;: leave vasculature: filter = 40 μm, magnified = 5 μm; stomata: filter = 2 μm, magnified = 5 μm. (c) Immunoblotting of protein extracts of two-week old seedlings of the indicated lines using α-GFP antibody for CES-YFP detection. Upper panel: autoradiogram, lower panel: coomassie brilliant blue staining (CBB) as a loading control. (d) Detection of SUMOylated CES in vivo. CES was immuno-precipitated from arabidopsis protoplasts transiently expressing 35S:CESS75A&S77A-Myc(6) (please note that CES tagged with Myc(6) is smaller as compared to CES tagged with YFP) using α-Myc antibody and detected by immunoblotting with an α-SUMO antibody.