FIG 5 .
MS/MS analysis of ubiquitinated NP revealed multiple ubiquitination sites. Tandem mass spectra of peptides derived from ubiquitinated NP show ubiquitin conjugation at different residues of ubiquitin with di-Gly modification. Ubiquitinated NP was separated as described for Fig. 3 and isolated using anti-HA–agarose from 293T cells transfected with NP-HA or Ub-myc, with or without CNOT4e-Flag. Protein samples were cut from the indicated protein bands (as for Fig. 3) and further purified (see Materials and Methods). Samples were analyzed by MS analysis. The Mascot search engine was used, with the criteria for searching ubiquitinated lysines marked as LeuArgGlyGly (KLRGG) or GlyGly (KGG). GG or LRGG modification was observed at the K31 (A), K77 (B), K87 (C), K91 (D), K113 (E), K184 (F), K227 (G), K229 (H), K273 (I), and K351 (J) residues.