4600 Nucleic Acids Research, 2006, Vol. 34, No. 16 Figure 2. Association of NOP132 with DDX18 and DDX47. (a) 293 cells were transfected with NOP132 and either FLAG-tagged DDX18 or FLAG-tagged DDX47. Cell lysates were prepared and used for immunoprecipitation with anti-FLAG as described (15). Total cell protein (2% input) (lanes 1–3). Immunoprecipitates (lanes 4–9). Proteins were detected with anti-NOP132N (upper panels), anti-FLAG (middle panels) or a RAN antibody against the nuclear protein RAN as a loading control (lower panels). Lane 4, control immunoprecipitate (NOP132 transfected); lane 5, control immunoprecipitate (NOP132 transfected) treated with ribonuclease; lane 6, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate; lane 7, immunoprecipitation with anti- FLAG of FLAG-tagged DDX47 lysate treated with ribonuclease; lane 8, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate; lane 9, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate treated with ribonuclease. (b) 293 cells were transfected with either FLAG-NOP132, FLAG-DDX18, or FLAG-DDX47. Silver-stained 10% SDS–PAGE gel of FLAG-NOP132-, FLAG-DDX47-, or FLAG-DDX18-associated complexes immunoprecipitated with anti-FLAG. Lane 1, molecular weight marker; lane 2, NOP132-associated proteins treated with ribonuclease; lane 3, NOP132- associated proteins; lane 4, DDX47-associated proteins treated with ribonuclease; lane 5, DDX47-associated proteins; lane 6, DDX18-associated proteins treated with ribonuclease; lane 7, DDX18-associated proteins; lane 8, control FLAG tag-associated proteins treated with ribonuclease; lane 9, control FLAG tag- associated proteins. Proteins which were identified by mass spectrometry are shown at the right of the gel image. (c) Baculovirus-produced NOP132 was mixed with GST (lane 1), GST-GRWD1 (lane 2), GST-DDX47 (lane 3), or GST-DDX18 (lane 4) bound to the glutathione-Sepharose-4B beads. Bound NOP132 was detected by western blotting using anti-NOP132N (upper panel). GST-fusion proteins stained with Coomassie brilliant blue are shown in the lower panel. Asterisks indicate the positions of the GST-fusion proteins.