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. 2011 Dec;85(24):12890–12900. doi: 10.1128/JVI.05628-11

Fig. 5.

Fig. 5.

E3 protein is differentially ubiquitinated depending on the integrity of a SIM domain. (A) MCF-7 cells were transfected with pCINEO-E3-WT in the presence or absence of pcDNA-His6-ubiquitin as indicated. Analysis of the protein extracts by Western blotting with an anti-E3 antibody 48 h after transfection was carried out. (B) MCF-7 cells were transfected as indicated above (left) or transfected with pcDNA-His6-ubiquitin and then infected with VACV for 6 h (right). Whole-protein extracts and histidine-tagged proteins purified by use of nickel columns were then analyzed by Western blotting with an anti-E3 antibody. Arrowheads refer to ubiquitinated E3. (C) MCF-7 cells were transfected with WT-E3- or E3L-ΔSIM-expressing plasmids in the presence or absence of pcDNA-His6-ubiquitin and treated with MG132 for 24 h. Analysis of the whole-protein extracts and the histidine-tagged proteins purified by use of nickel columns was then carried out by Western blotting with an anti-E3 antibody.