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. 2017 Dec 20;7:17936. doi: 10.1038/s41598-017-18256-3

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Schematic primary sequences of the STIM2 and STIM2-UI region, binding assay of GST-fused STIM1-UI protein with a triad sample, and co-immunoprecipitation of full-length STIM2 with SERCA1a. (a) The position of each domain in STIM2 is presented according to previous reports: the overall diagram^92,93, and CAD/SOAR⁹⁴. Numbers indicate the sequence of amino acids. UI means unidentified region. S, signal peptide; cEF, canonical EF-hand; hEF, non-functional hidden EF-hand; SAM, sterile α-motif; T, transmembrane domain; C, coiled-coil domain; CAD/SOAR, Ca²⁺ release-activated Ca²⁺ (CRAC)-activating domain/STIM1-Orai1-activating region; P, proline/serine-rich domain; K, lysine-rich domain. (b) Immobilized GST-fused STIM2-UI proteins on GST beads were separated on a 10% SDS-PAGE gel, and the gel was subjected to immunoblot with anti-GST antibody. GST or GST-fused STIM2-UI proteins are indicated by asterisks. (c) The bound proteins in the binding assay of GST-fused STIM2-UI protein with the triad sample from rabbit skeletal muscle were separated on a 10% SDS-PAGE gel, and the gel was stained with Coomassie Blue. GST was used as a negative control. GST-fused STIM2-UI proteins are indicated by an asterisk. The eight proteins that were bound to the GST-fused STIM2-UI protein are indicated on the right side of the figure (bands 1 to 8). (d) The triad sample obtained from rabbit skeletal muscle (30 μg of total proteins) was subjected to a co-immunoprecipitation assay with anti-STIM2 antibody, and the immunoprecipitant was subjected to immunoblot analysis with anti-STIM2 or anti-SERCA1a antibodies. Triad sample indicates a simple immunoblot of the triad sample. Without Ab indicates a reaction without anti-STIM2 antibody. Three independent experiments were conducted. IB, IP, or Ab means immunoblot, immunoprecipitation, or antibody, respectively. The immunoblot data were cropped from the immunoblot images of different gels and were grouped. The full-length blots are presented in Supplementary Fig. 4. SERCA1a was successfully co-immunoprecipitated with full-length STIM2.