Expression and Characterization of STIM1 Variants

Full-length cDNAs of human STIM1 was subcloned into pCMV6-XL5 (Origene)^{37 (link)} with the insertion of EGFP, YFP or CFP between two additional NarI sites introduced immediately after residue N39. STIM1 mutant constructs were subsequently made by using the QuikChange Lightning site-directed mutagenesis kit (Agilent). For STIM1-CFP and STIM1-YFP constructs, human STIM1 was inserted into pECFP-N1 or pEYFP-N1 between XhoI and BamHI. Truncated STIM1-CFP and STIM1-YFP variants were prepared by standard PCR and ligation. pCDNA3.1(+)-mCherry-ORAI1 was made by inserting mCherry between BamHI-EcoRI restriction sites and human ORAI1 between EcoRI-XhoI sites (Life Technologies). NFAT1_1–460-GFP^{38 (link)}, mCherry-CAD^{9 (link)}, YFP-SOAR^{10 (link)} and YFP-D-SOAR (tandem SOAR domain)^{39 (link),40 (link)} were obtained from Addgene or generated as previously described.

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Ma G., Wei M., He L., Liu C., Wu B., Zhang S.L., Jing J., Liang X., Senes A., Tan P., Li S., Sun A., Bi Y., Zhong L., Si H., Shen Y., Li M., Lee M.S., Zhou W., Wang J., Wang Y, & Zhou Y. (2015). Inside-out Ca2+ signalling prompted by STIM1 conformational switch. Nature Communications, 6, 7826.

Publication 2015

pCMV6-XL5 QuikChange Lightning site-directed mutagenesis kit mCherry-CAD9 YFP-SOAR10 YFP-D-SOAR

Cdnas Ecori Human Ligation Site directed mutagenesis Stim1

Corresponding Organization :

Other organizations : Texas A&M Health Science Center, Texas A&M University, Beijing Normal University, Hefei Institutes of Physical Science, High Magnetic Field Laboratory, Chinese Academy of Sciences, University of Wisconsin–Madison, Nankai University, Shandong University, University of Michigan–Ann Arbor, University of Science and Technology of China

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Variable analysis

independent variables

Insertion of EGFP, YFP or CFP between two additional NarI sites introduced immediately after residue N39 of STIM1
STIM1 mutant constructs made using site-directed mutagenesis
Truncated STIM1-CFP and STIM1-YFP variants prepared by standard PCR and ligation

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive controls: NFAT1_1-460-GFP, mCherry-CAD, YFP-SOAR, YFP-D-SOAR
Negative control: Not specified

Annotations

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