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

Leica confocal software (LCS Lite) was used to quantify the numbers of nucleolar and nuclear (excluding nucleoli) eGFP fusion proteins in fixed transfected HeLa cells. All the images (10246768 pixels) were obtained using a TCM-SL confocal microscope (LEICA) with a 636 objective lens. Confocal images were quantified using settings where the intensity of eGFP fluorescence in the analyzed transfectans was linear and ranged from 10 to 160 pixel values [42] . Usually, 10 sections encompassing an entire cell were taken at 1.0-mm interval. To examine the localization of eGFP and eGFP fusion proteins, a square with an area of 50 pixels was used to measure the mean intensities of three different regions in two compartments -the nucleolus and the nucleus (excluding the nucleolus) -of four representative cells from each of three transfections. Thus, the twelve representative cells yielded a total of 36 areas from the nucleolus and 36 areas from the nucleus. Relative nucleolar and nuclear localization is reported as the mean of the percentage of the relative intensity of GFP fluorescence within a cell.