Figure 6. Deneddylation by yeast and mammalian CSN complexes that lacked the MPN⁻ domain on yeast or mammalian cullin substrates.

A: The rubylated Cdc53 in Δcsn5 extract was used as a substrate to test the deneddylation activity of Flag-purified mammalian complexes. Flag-S6CD, Flag-S6MPN, or empty vector were expressed in HeLa cells, isolated via the Flag beads, and eluted with the Flag peptide. The proteins were immunoblotted with indicated antibodies. Yeast cell extracts of wild type, Δrub1 and Δcsn5 strains served as size markers for Cdc53 rubylated and un-rubylated forms. Asterisk indicates IgG background. B: Purified CSN complexes from yeast (ScCSN) or mammalian cells (CSN^PS) were tested for the deneddylation activity on human Cul4a-Nedd8 conjugates in CSN-depleted HeLa cell extract. Δcsn5 extract from yeast was used as a negative control. UT, untreated cell extract.