(A) TAP-tagged assembly factor Nog2 was used as the bait to purify late nuclear pre-60S particles. Protein composition was assayed by SDS-PAGE, followed by silver staining, when wild-type L8 or the L8Δ1-70 mutant protein was expressed as the sole version of L8. (B) The levels of indicated assembly factors and r-proteins were tested by Western blots. As expected, Ebp2 and Has1 were not detected in Nog2-associated particles; these assembly factors mostly dissociate before Nog2 assembles into pre-ribosomes. (C) Changes in levels of large ribosomal subunit assembly factors in the L8Δ1-70 mutant compared to wild-type L8 were determined by semiquantitative iTRAQ analysis. Relative levels of each protein were normalized to the ratio of the Nog2 bait protein, and results were reported on a log2 scale. The blue and red bars correspond to the ratio of each assembly factor in mutant versus wild-type (mutant: WT) for duplicate experiments. The dashed lines indicate arbitrarily chosen cutoffs where the average ratio of duplicated experiments increases to greater than three, or decreases to less than five.
