Cloning and Mutagenesis of RLTPR

The C-terminal Myc/DDK-tagged pCMV6 empty vector and the human RLTPR expression vector were purchased from OriGene (RC217662). Constructs carrying mutant alleles were generated from this plasmid by direct mutagenesis with a site-directed mutagenesis kit (QuickChange II XL; Agilent Technologies), according the manufacturer’s instructions. For coimmunoprecipitation experiments, full-length RLTPR WT cDNA was subcloned into the V5/His-tagged pcDNA3.1 plasmid using the directional TOPO expression kit (Thermo Fisher Scientific). HEK293T cells were transiently transfected with the various constructs, using the Lipofectamine LTX kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The 3D structure of the CARMIL LRR region was previously described (Zwolak et al., 2013 (link)). The 3D structure picture and mutations were displayed using Protein Workshop 4.2.0 (Moreland et al., 2005 (link)).

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Wang Y., Ma C.S., Ling Y., Bousfiha A., Camcioglu Y., Jacquot S., Payne K., Crestani E., Roncagalli R., Belkadi A., Kerner G., Lorenzo L., Deswarte C., Chrabieh M., Patin E., Vincent Q.B., Müller-Fleckenstein I., Fleckenstein B., Ailal F., Quintana-Murci L., Fraitag S., Alyanakian M.A., Leruez-Ville M., Picard C., Puel A., Bustamante J., Boisson-Dupuis S., Malissen M., Malissen B., Abel L., Hovnanian A., Notarangelo L.D., Jouanguy E., Tangye S.G., Béziat V, & Casanova J.L. (2016). Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations. The Journal of Experimental Medicine, 213(11), 2413-2435.

Publication 2016

QuickChange II XL directional TOPO expression kit Lipofectamine LTX kit

Alleles Cdna Cells Coimmunoprecipitation Human Lipofectamine Mutagenesis Mutations Plasmid Protein Site directed mutagenesis Topo Vector

Corresponding Organization : Institut des Maladies Génétiques Imagine

Other organizations : Garvan Institute of Medical Research, St Vincent's Clinic, UNSW Sydney, University of Hassan II Casablanca, Istanbul University, Institute for Research and Innovation in Biomedicine, Université de Rouen Normandie, Boston Children's Hospital, Centre d’Immunologie de Marseille-Luminy, Institut Pasteur, Centre National de la Recherche Scientifique, Friedrich-Alexander-Universität Erlangen-Nürnberg, Assistance Publique – Hôpitaux de Paris, Sorbonne Paris Cité, Délégation Paris 5, Rockefeller University, Human Genetic of Infectious Diseases, Harvard Stem Cell Institute, Harvard University, Howard Hughes Medical Institute

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Variable analysis

independent variables

Mutant alleles of RLTPR

dependent variables

Coimmunoprecipitation of RLTPR

control variables

Wild-type RLTPR

controls

Positive control: Wild-type RLTPR
Negative control: Not explicitly mentioned

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