Authors: <div class="autor_fcen" id="8534">Thomas, M.G.</div>; <div class="autor_fcen" id="5196">Luchelli, L.</div>; Pascual, M.; Gottifredi, V.; <div class="autor_fcen" id="982">Boccaccio, G.L.</div>
Publication Date: 2012.
The p53 tumor suppressor protein is an important regulator of cell proliferation and apoptosis. p53 can be found in the nucleus and in the cytosol, and the subcellular location is key to control p53 function. In this work, we found that a widely used monoclonal antibody against p53, termed Pab 1801 (Pan antibody 1801) yields a remarkable punctate signal in the cytoplasm of several cell lines of human origin. Surprisingly, these puncta were also observed in two independent p53-null cell lines. Moreover, the foci stained with the Pab 1801 were present in rat cells, although Pab 1801 recognizes an epitope that is not conserved in rodent p53. In contrast, the Pab 1801 nuclear staining corresponded to genuine p53, as it was upregulated by p53-stimulating drugs and absent in p53-null cells. We identified the Pab 1801 cytoplasmic puncta as P Bodies (PBs), which are involved in mRNA regulation. We found that, in several cell lines, including U2OS, WI38, SK-N-SH and HCT116, the Pab 1801 puncta strictly colocalize with PBs identified with specific antibodies against the PB components Hedls, Dcp1a, Xrn1 or Rck/p54. PBs are highly dynamic and accordingly, the Pab 1801 puncta vanished when PBs dissolved upon treatment with cycloheximide, a drug that causes polysome stabilization and PB disruption. In addition, the knockdown of specific PB components that affect PB integrity simultaneously caused PB dissolution and the disappearance of the Pab 1801 puncta. Our results reveal a strong cross-reactivity of the Pab 1801 with unknown PB component(s). This was observed upon distinct immunostaining protocols, thus meaning a major limitation on the use of this antibody for p53 imaging in the cytoplasm of most cell types of human or rodent origin. © 2012 Thomas et al.
Author affiliation: Thomas, M.G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Author affiliation: Luchelli, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Author affiliation: Boccaccio, G.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Keywords: cell marker; cycloheximide; decapping enzyme 1a; decapping enzyme 1b; decapping enzyme 2; epitope; exoribonuclease; exoribonuclease 1; messenger RNA; monoclonal antibody; pantropic antibody 1801; protein 4ET; protein Hedls; protein p53; protein p54; small interfering RNA; unclassified drug; epitope; protein p53; TP53 protein, human; animal cell; animal tissue; article; cell component; cell disruption; cell line; cell stimulation; cell strain HCT116; cellular distribution; concentration (parameters); controlled study; cross reaction; dissolution; Drosophila; embryo; fetus; gene control; gene silencing; genetic transfection; human; human cell; image analysis; immunohistochemistry; intracellular signaling; nonhuman; polysome; processing body; protein analysis; protein localization; rat; upregulation; animal; antibody specificity; chemistry; cytoplasm; immunology; metabolism; Sprague Dawley rat; tumor cell line; Rattus; Rodentia; Animals; Antibodies, Monoclonal, Murine-Derived; Antibody Specificity; Cell Line, Tumor; Cytoplasm; Epitopes; Humans; Immunohistochemistry; Rats; Rats, Sprague-Dawley; Tumor Suppressor Protein p53.
Repository: Biblioteca Digital (UBA-FCEN). Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales
Publication Date: 2017.
Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, highresolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such largescale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.
Author affiliation: Hansen, Christian Skjødt. Technical University of Denmark; Dinamarca
Author affiliation: Østerbye, Thomas. Universidad de Copenhagen; Dinamarca
Author affiliation: Marcatili, Paolo. Technical University of Denmark; Dinamarca
Author affiliation: Lund, Ole. Technical University of Denmark; Dinamarca
Author affiliation: Buus, Søren. Universidad de Copenhagen; Dinamarca
Author affiliation: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas