Fig. 1.

Association of p32 with several proteins involved in ribosome biogenesis. A, Equivalent amounts of cytoplasmic (Cyto) or nuclear (Nu) extract prepared from mock- or p32-FLAG (p32)-transfected 293EBNA cells were analyzed by immunoblotting (IB) with anti-GAPDH as a marker of the cytoplasm, anti-Lamin B as a marker of the nucleus, and anti-FLAG for the detection of the expressed p32-FLAG. B, Nuclear extracts prepared from 293EBNA cells transfected with a plasmid containing a FLAG- (mock) or p32-FLAG-encoded DNA fragment (p32) were subjected to immunoprecipitation with anti-FLAG-fixed beads. The isolated proteins as well as an aliquot of the nuclear extract of mock- or p32-FLAG-transfected cells (input, 5 μg) were analyzed by 10% SDS-PAGE and visualized by silver staining. The asterisk indicates p32-FLAG used as bait. The molecular masses (kDa) are indicated at the left side of the figure. C, Association of p32-FLAG with the proteins identified by LC-MS/MS analysis and presence of ASF/SF2 was confirmed by immunoblot analysis with the indicated antibodies. D, The presence of known pre-rRNA processing factors that were not identified by LC-MS/MS analysis was examined by immunoblot analysis of the isolated p32-FLAG-associated proteins with the indicated antibodies.