Orai1 Construct Cloning and Mutation

N-terminally tagged Orai1 constructs (accession number NM_032790.3) were cloned into the SalI and SmaI restriction sites of pECFP-C1 and pEYFP-C1 expression vectors (Clontech). GFP-TFEB and GFP-MITF were purchased from Addgene and subcloned into peCFP vectors. hOrai1 Δ1-64, N223A, and cysteine-free (C126V, C143V, C195V) constructs were used for crosslinking as described in (12 (link)). Point mutations in Orai1 constructs were introduced using the QuikChange site-directed mutagenesis kit (Stratagene). The integrity of all resulting mutants was confirmed by sequence analysis (Eurofins Genomics).

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Frischauf I., Litviňuková M., Schober R., Zayats V., Svobodová B., Bonhenry D., Lunz V., Cappello S., Tociu L., Reha D., Stallinger A., Hochreiter A., Pammer T., Butorac C., Muik M., Groschner K., Bogeski I., Ettrich R.H., Romanin C, & Schindl R. (2017). Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription. Science signaling, 10(507), eaao0358.

Publication 2017

pECFP-C1 pEYFP-C1 expression vectors GFP-TFEB

Cysteine Mitf Point mutations Sequence analysis Site directed mutagenesis Smai Tfeb Vectors

Corresponding Organization : Medical University of Graz

Other organizations : Johannes Kepler University of Linz, Max Delbrück Center, University of Warsaw, Czech Academy of Sciences, Czech Academy of Sciences, Institute of Microbiology, University of Göttingen, Saarland University, University of Chicago, University of South Bohemia in České Budějovice, University of Graz, Paracelsus Medical University

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Variable analysis

independent variables

N-terminally tagged Orai1 constructs (accession number NM_032790.3)
HOrai1 Δ1-64
HOrai1 N223A
HOrai1 cysteine-free (C126V, C143V, C195V) constructs

dependent variables

Not explicitly mentioned

control variables

ECFP-C1 and EYFP-C1 expression vectors (Clontech)
GFP-TFEB and GFP-MITF constructs

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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