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. 2013 Apr 15;288(21):14716–14726. doi: 10.1074/jbc.M112.448258

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 9. — Overexpression of DCN1 alleviates CIF-mediated inhibition of neddylation but does not restore NEDD8-dependent cell proliferation. A, the rub1Δ yeast strains expressing NEDD8, galactose-inducible CIF (MPY231), and either ADH-HA-DCN1 (DCN1 overexpression (O/E)) or a vector control (none) were grown to late log phase. Galactose was added to 2%, and samples taken at the indicated times were analyzed by immunoblotting with the indicated antibodies. B, lysates from rub1Δ yeast strains (MPY156) coexpressing either NEDD8 (WT) or NEDD8 Q40E and ADH-HA-DCN1 or a vector control were analyzed by immunoblotting with the indicated antibodies. C, the effect of Dcn1 on NEDD8-dependent cell growth was evaluated in yeast strains expressing galactose-inducible CIF (WT or C109S, top panel) or NEDD8 (Q40E or WT; bottom panel). Serial dilutions were spotted onto glucose- or galactose-containing medium for inducible CIF expression and medium without or with 5-FOA for NEDD8 conditional growth.