Construction of V5-Tagged and SUMOylation Mutant HuR Plasmids
The full length cDNA of WT mouse HuR was purchased from the German Resource Center for Genome Research (RZPD). The V5-HuR WT plasmid was constructed by PCR using a 5′ oligonucleotide containing the V5 tag sequence and being subcloned into a pcDNA3.3-TOPO vector (K830001, Invitrogen).66 (link) SUMOylation sites on HuR were predicted experimentally by mutation of lysine residues into arginine. The SUMOylation mutant HuR plasmid constructs were created using the QuickChange site-directed mutagenesis kit (200518, Stratagene), according to the manufacturer’s instructions, with two complementary oligonucleotides and with pcDNA3.3-TOPO-V5-HuR WT plasmid as template. Products were sequenced by STAB vida. Plasmid DNA was purified after bacterial transformation and amplification, using NucleoBond Xtra Midi Plus kit (740412, Macherey-Nagel), by following the manufacturer’s instructions.
Lachiondo-Ortega S., Rejano-Gordillo C.M., Simon J., Lopitz-Otsoa F., Delgado T.C., Mazan-Mamczarz K., Goikoetxea-Usandizaga N., Estefanía Zapata-Pavas L., Garcıía-del Río A., Guerra P., Peña-Sanfélix P., Hermán-Sánchez N., Al-Abdulla R., Fernandez-Rodríguez C., Azkargorta M., Velázquez-Cruz A., Guyon J., Martín C., Zalamea J.D., Egia-Mendikute L., Sanz-Parra A., Serrano-Maciá M., González-Recio I., Gonzalez-Lopez M., Martínez-Cruz L.A., Pontisso P., Aransay A.M., Barrio R., Sutherland J.D., Abrescia N.G., Elortza F., Lujambio A., Banales J.M., Luque R.M., Gahete M.D., Palazón A., Avila M.A., Marin J.J., De S., Daubon T., Díaz-Quintana A., Díaz-Moreno I., Gorospe M., Rodríguez M.S, & Martínez-Chantar M.L. (2024). SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer. Cell reports, 43(3), 113924.
Corresponding Organization : Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas
Other organizations :
Universidad de Extremadura, Consejo Superior de Investigaciones Científicas, University of the Basque Country, Institute on Aging, National Institutes of Health, National Institute on Aging, University of Padua, University of Córdoba, Instituto Maimónides de Investigación Biomédica de Córdoba, Universitätsmedizin Greifswald, Universitat de Miguel Hernández d'Elx, Instituto de Investigaciones Químicas, Universidad de Sevilla, Centro de Investigaciones Científicas Isla de la Cartuja, Centre Hospitalier Universitaire de Bordeaux, Inserm, Université de Bordeaux, Ikerbasque, Icahn School of Medicine at Mount Sinai, Biogipuzkoa Health Research Institute, Universidad de Navarra, Navarre Institute of Health Research, Instituto de Investigación Biomédica de Salamanca, Universidad de Salamanca, Centre National de la Recherche Scientifique, Université Toulouse III - Paul Sabatier, Laboratoire de Chimie de Coordination
Mutation of lysine residues into arginine on HuR protein
dependent variables
SUMOylation of HuR protein
control variables
PcDNA3.3-TOPO-V5-HuR WT plasmid as template for site-directed mutagenesis
controls
PcDNA3.3-TOPO-V5-HuR WT plasmid as a positive control
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