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Conjugation of mammalian NEDD8 in yeast. The total conjugation pattern of HANEDD8 (compared with HARUB1) in yeast was determined by Western blotting with HA-specific antibodies. The lower band corresponds to free RUB1 (first lane) or NEDD8 (other lanes). NEDD8 conjugates are virtually absent in ula1, uba3, and ubc12 mutants defective in RUB1 conjugation. The immunoreactive 30-kDa protein is a crossreacting yeast protein.
