Figure 2.

NAP57 is a specific component of box H/ACA snoRNPs, whereas Nopp140 associates with both box H/ACA and box C/D snoRNPs. (A) Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by amido black staining. Nopp140 (lanes 2 and 3) and NAP57 (lanes 4 and 5) were precipitated from rat liver nuclear extracts (lane 1) with peptide antibodies in the absence (lanes 2 and 4) and presence (lanes 3 and 5) of competing peptide. Proteins that precipitated exclusively in the absence of competing peptide are listed on the right. NAP65 is indicated by a dot because it stains very poorly by amido black. (B) The membrane in A was probed consecutively with antibodies specific for the antigens listed on the left, and the antibody reactivity was detected by ECL. All of the films showing the ECL results of the individual antibodies were overlaid and are displayed simultaneously. (C) Box H/ACA (top two panels) and box C/D (bottom two panels) snoRNAs were immunoprecipitated from whole cell extracts (lane 1), and their RT-PCR products were separated on agarose gels and detected by ethidium bromide. NAP57 (lanes 2 and 3) and Nopp140 (lanes 4 and 5) were precipitated in the absence (lanes 2 and 4) and presence (lanes 3 and 5) of competing peptide, total RNAs were extracted, and the snoRNAs were amplified by RT-PCR with primers specific for the snoRNAs indicated on the left. To improve the visibility of the precipitated box C/D snoRNA RT-PCR products, the contrast of lanes 2–5 of the bottom two panels was selectively increased.