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Fig. 4 | BMC Plant Biology

Fig. 4

From: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

Fig. 4

Wheat germ-based in vitro ubiquitination analysis of RING proteins showed high molecular smears. a At4g11680 and its corresponding RING mutant were expressed with biotin tag and analysed as a representative protein in the presence or absence of FLAG-Ub, E1, and/or AtUBC8 without tag for the ubiquitination activity. Replacement of the third metal ligand, Cys to Ser, caused reduced smear upon blotting against FLAG-Ub with anti-FLAG-HRP antibody. Absence of the E1 or AtUBC8 from the reaction did not abolish the high molecular smear. b Three other RING proteins At1g80400, At2g22120, and At1g11020, and their RING mutants together with At4g11680, were analysed in the presence of FLAG-Ub. The four proteins with RING mutants showed significantly reduced activity upon blotting with anti-FLAG-HRP antibody, whereas the proteins with intact RING domains showed activity without the addition of E1 or E2. The side arrow refers to the free FLAG-Ub that migrated to the bottom of the gel

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