Journal: Molecular and Cellular Biology
Article Title: Naf1p, an Essential Nucleoplasmic Factor Specifically Required for Accumulation of Box H/ACA Small Nucleolar RNPs †
Figure Lengend Snippet: Analysis of the interactions between Naf1p-ZZ and H/ACA snoRNP components. Total extracts were produced in native conditions from yeast cells expressing either free ZZ tag (lanes 1 to 3), ZZ-Nop1p (lanes 4 to 6), Cbf5p-ZZ (lanes 7 to 9), or Naf1p-ZZ (lanes 10 to 12). Immunoprecipitation experiments were carried out using IgG-Sepharose beads in a buffer containing either 150 mM (lanes 2, 5, 8, and 11) or 500 mM (lanes 3, 6, 9, and 12) KOAc. After precipitation and being washed, beads were separated in two equal fractions. (A) Western blot analysis. Beads from one set of fractions were resuspended in protein-denaturing buffer. Aliquots of the resulting supernatants (lanes 2, 3, 5, 6, 8, 9, 11, and 12) and 1/20 of the corresponding amount of total proteins from the input extract (T and lanes 1, 4, 7, and 10) were submitted to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to cellulose membranes. Tagged proteins were detected by use of rabbit PAP, Nsr1p by a monoclonal antibody, and Nop1p, Gar1p, Nhp2p, and Nop10p by polyclonal sera. Northern blot (B) and analysis (C) of [ 32 P]pCp-labeled RNAs. RNAs retained on the beads of the second set of fractions were purified. Aliquots of precipitated RNAs (lanes 2, 3, 5, 6, 8, 9, 11, and 12) and 1/10 of the corresponding amount of total RNAs from the input extract (T and lanes 1, 4, 7, and 10) were submitted to denaturing 6% polyacrylamide gel electrophoresis. In panel B, separated RNAs were transferred to nylon membranes, and various H/ACA and C/D snoRNAs were detected by hybridization with specific oligonucleotide probes. Phosphorimager scans of the Northern blots were used to quantify snoRNA levels. The amounts of RNAs precipitated, expressed as percentages of input RNA levels, are indicated in the table on the right. In panel C, RNAs were 3′ end labeled with [ 32 P]pCp and separated on a 6% sequencing gel. M, molecular weight markers (pBR322 digested with Hae III and Taq I). Positions of RNAs inferred from their size are indicated.
Article Snippet: Naf1p-ZZ and Cbf5p-TAP were detected using rabbit PAP (Dako) diluted 10,000-fold.
Techniques: Produced, Expressing, Immunoprecipitation, Western Blot, Polyacrylamide Gel Electrophoresis, Northern Blot, Labeling, Purification, Hybridization, Sequencing, Molecular Weight