Plasmid Construction and Antibody Details

Plasmids expressing cMyc-tagged mouse SEPN1 (with A1284T/C conversion of the TGA selenocysteine codon into Cys to increase expression) and cMyc-tagged human Desmin were constructed in a CMV promoter-derived mammalian expression vector.
Expression plasmids encoding N-terminally FLAG-tagged human SEPN1 and the mutants SEPN1 SS (C427S, U428S) and SEPN1 CC (U428C) were constructed in pSel-Express vector (a kind gift from Vladimir Gladyshev) [16 ].
The primary antibodies used were: anti-VDAC2, anti-Cyt C, anti-Calnexin, anti-SERCA2, anti-RyR, anti-cMyc, and anti-PDI from Abcam; anti-Flag, anti-β-tubulin, and anti-Sigma 1-R from Sigma-Aldrich; anti-IP3R3 and Cyt c from BD Biosciences; anti-Tom40 from Santa Cruz, and homemade rabbit anti-SEPN1 [21 (link)]. Secondary antibodies Alexa Fluor 488 and 594 were from Life Technologies.

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Filipe A., Chernorudskiy A., Arbogast S., Varone E., Villar-Quiles R.N., Pozzer D., Moulin M., Fumagalli S., Cabet E., Dudhal S., De Simoni M.G., Denis R., Vadrot N., Dill C., Giovarelli M., Szweda L., De Palma C., Pinton P., Giorgi C., Viscomi C., Clementi E., Missiroli S., Boncompagni S., Zito E, & Ferreiro A. (2020). Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy. Cell Death and Differentiation, 28(1), 123-138.

Publication 2020

anti-VDAC2 anti-Cyt C anti-Calnexin anti-SERCA2 anti-RyR anti-cMyc anti-PDI anti-Flag anti-β-tubulin anti-Sigma 1-R anti-Tom40 Alexa Fluor 488

Alexa fluor 488 Antibodies Calnexin Desmin Human Mammalian Mouse Plasmids Rabbit Selenocysteine Serca2 Tga codon Tubulin Vdac2 Vector

Corresponding Organization : Assistance Publique – Hôpitaux de Paris

Other organizations : Unit of Functional and Adaptive Biology, Université Paris Cité, Centre National de la Recherche Scientifique, Mario Negri Institute for Pharmacological Research, Istituti di Ricovero e Cura a Carattere Scientifico, The University of Texas Southwestern Medical Center, University of Ferrara, University of Padua, University of Chieti-Pescara

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Variable analysis

independent variables

Expression plasmids encoding N-terminally FLAG-tagged human SEPN1 and the mutants SEPN1 SS (C427S, U428S) and SEPN1 CC (U428C)

dependent variables

Not explicitly mentioned

control variables

Plasmids expressing cMyc-tagged mouse SEPN1 (with A1284T/C conversion of the TGA selenocysteine codon into Cys to increase expression)
Plasmids expressing cMyc-tagged human Desmin

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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