Journal:

Article Title: Conserved Composition of Mammalian Box H/ACA and Box C/D Small Nucleolar Ribonucleoprotein Particles and Their Interaction with the Common Factor Nopp140

doi:

Figure Lengend Snippet: Transient expression of the dominant negative Nopp140 carboxyl terminus chases snoRNPs out of the nucleolus (A) and arrests RNA pol I transcription (B) without affecting the localization of RNA pol I (C). Cells were transfected with the HA-tagged carboxyl terminus of Nopp140, and subsequently endogenous GAR1 (a) and RNA pol I (c) were labeled by indirect double immunofluorescence or run-on transcription was detected by BrUTP incorporation and visualization with anti-BrdU antibodies (b). The transfected cells (solid arrows) were identified with anti-HA antibodies (a′ and c′) or by the aberrant nucleoplasmic distribution of endogenous Nopp140 detected with Nopp140-specifc antibodies (b′). All nucleoli can be clearly identified in the phase-contrast images (a′′, b′′, and c′′). One nucleolus of a transfected and an untransfected cell is marked in each panel (small open arrows). Bar, 10 μm.

Article Snippet: The cells were postfixed with 2% paraformaldehyde for 15 min, and 5-bromo-uridine-triphosphate (BrUTP) (Sigma Chemical) incorporation was detected with a mouse anti-5-bromo-2′-deoxyuridine (BrdU) mAb, F(ab′) 2 fragments conjugated with FLUOS (Boehringer Mannheim) at a dilution of 1:5.

Techniques: Expressing, Dominant Negative Mutation, Transfection, Labeling, Immunofluorescence