Nucleic Acids Research, 2006, Vol. 34, No. 16 4597 Figure 1. Protein components of immunoprecipitated FLAG-NOP132-, FLAG-DDX47-, and FLAG-DDX18-associated complexes. (a) Silver-stained SDS–PAGE gels of FLAG-NOP132-associated complexes immunoprecipitated with anti-FLAG from cells expressing FLAG-tagged NOP132. Lane 1, control immunoprecipitate (Mock) from untransfected 293EBNA cells; lane 2, 293EBNA cells expressing FLAG-tagged NOP132 (11% SDS–PAGE gel); lane 3, 293 cells expressing FLAG-tagged NOP132 (lysis by sonication, 10% SDS–PAGE gel)]. Molecular weight markers are indicated at the left. Because there were so many protein bands in the gels, the gel slices contained multiple proteins. The protein bands identified by mass spectrometric analysis after in-gel digestion of protein bands with protease are indicated on the right. Proteins that were also identified in the FLAG-DDX47-associated complex are underlined. (b) Silver- stained 10% SDS–PAGE gel of FLAG-DDX47-associated complexes immunoprecipitated with anti-FLAG after expression of FLAG-tagged full-length DDX47 in 293 cells. Molecular weight markers are indicated at the left. The protein bands identified by mass spectrometric analysis after in-gel digestion of protein bands with protease are indicated on the right. Proteins that were also identified in the FLAG-NOP132-associated complex are underlined. (c) Silver-stained 10% SDS–PAGE gel of FLAG-DDX18-associated complexes immunoprecipitated with anti-FLAG after expression of FLAG-tagged full-length DDX18 in 293 cells. Molecular weight markers are indicated at the left. The protein bands identified by mass spectrometric analysis after in-gel digestion of protein bands with protease are indicated on the right. Proteins that were also identified in FLAG-NOP132- and FLAG-DDX47-associated complexes are underlined and in bold, respectively. immunoprecipitate NOP132-associated proteins from human 293EBNA cells expressing FLAG-tagged NOP132. Silver- stained SDS–PAGE gels of the immunoprecipitated fraction indicated that the NOP132 complexes contained many pro- teins spanning a wide range of molecular weights (Figure 1a, lane 2). In contrast, only three protein bands were apparent in a mock immunoprecipitate prepared from untransfected con- trol cells (Figure 1, lane 1). These results suggest that many of the proteins immunoprecipitated from FLAG-NOP132- transfected cells represent NOP132-associated proteins. The protein components of the NOP132 complexes were identiﬁed using mass spectrometry. Protein identiﬁcation was ﬁrst performed for individual bands excised from SDS– PAGE gels using in-gel digestion and matrix-assisted laser desorption ionization-time of ﬂight mass spectroscopy. This approach identiﬁed RNA helicase DEAD/H (Asp-Glu-Ala- Asp/His) box polypeptide 47 (DDX47), ribosomal protein L3 (RPL3), glutamate-rich WD repeat protein (GRWD1), and KIAA0539 [NOP254, a possible human ortholog to yeast Npa1p (31)] as candidates for NOP132-associated proteins (Figure 1a, lane 2). A nanoscale LC-MS/MS system con- ﬁrmed the presence of these proteins in the FLAG immuno- precipitates (Supplementary Table 1). A similar identiﬁcation experiment was performed to isolate more complex- associated proteins using 293 cells as recipient (Figure 1a, lane 3). We found that most of the NOP132 was resistant to solubilization with our immunoprecipitation buffer, but could be more completely solubilized upon brief soni- cation. Thus, we brieﬂy sonicated NOP132-transfected 293 cells and solubilized FLAG-tagged NOP132 efﬁciently (Figure 1a, lane 3). These experiments also identiﬁed nucleolin/NCL (yeast ortholog Nsr1), B23/NPM1, ﬁbrillarin/ FBL (Nop1), Nop5/Nop58, BRIX/BXDC2 (Brx1), putative nucleotide-binding protein estradiol-induced nuclear GTPase/ GNL3 (Nug1), RNA helicase Gu/DDX21, DHX9, DDX18 (Has1), many ribosomal proteins from the large and small subunits, and other non-ribosomal proteins, most of which were found in previously characterized human pre-ribosomal ribonucleoprotein (rRNP) complexes, as candidates for NOP132-associated proteins (Figure 1a; Tables 1 and 2;