NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

For indirect immunofluorescence staining, cells grown on coverslips were washed with PBS and fixed in cold acetone for 10 min at 220uC (anti-NOA36, anti-UBF and anti-Flag antibodies) or 4% paraformaldehyde before permeabilization with 0.1% Triton X-100 (eGFP transfected cells). Cells were then washed with PBS and incubated with primary antibodies diluted in PBS (1:100 of anti-NOA36; 1:100 of anti-UBF or 1:500 of anti-Flag M2 (SIGMA)) at 37uC for 45 min. Cells were then washed with PBS for 30 min at room temperature and incubated with Alexa fluor 488, 555 (Molecular Probes), or Cy3 (Jackson ImmunoResearch)-labelled secondary antibodies at 37uC for 45 min. Finally, cells were washed twice in PBS and mounted in PBS-glycerol containing DAPI at 0.1 mg/ml, for DNA staining. A Zeiss Axiophot microscope equipped with a 636NA 1.3 oilimmersion objective was routinely used. Images were taken using a SPOT Camera (Diagnostic Instruments Inc.) and processed using Adobe Photoshop software.