Abstract
The characterization of protein-protein interactions through methods such as co-immunoprecipitation, followed by Western blot analysis, is a crucial step in the understanding of protein functions and the biology of the cell. Since the discovery of ORAI and STIM proteins as component of store-operated channel (SOC), overexpressing systems have been used to demonstrate how ORAI and STIM can associate with physiological and pathological conditions. Here we describe a protocol allowing endogenous studies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Feske S, Gwack Y, Prakriya M et al (2006) A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441:179–185. https://doi.org/10.1038/nature04702
Liou J, Kim ML, Heo WD et al (2005) STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr Biol 15:1235–1241. https://doi.org/10.1016/j.cub.2005.05.055
Wu MM, Buchanan J, Luik RM, Lewis RS (2006) Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J Cell Biol 174:803–813. https://doi.org/10.1083/jcb.200604014
Smyth JT, Hwang S-Y, Tomita T et al (2010) Activation and regulation of store-operated calcium entry. J Cell Mol Med 14:2337–2349. https://doi.org/10.1111/j.1582-4934.2010.01168.x
Putney JW (2011) Origins of the concept of store-operated calcium entry. Front Biosci (Schol Ed) 3:980–984
Yang X, Jin H, Cai X, et al Structural and mechanistic insights into the activation of Stromal interaction molecule 1 (STIM1). Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5657–62. https://doi.org/10.1073/pnas.1118947109 Epub 2012 Mar 26.
Motiani RK, Abdullaev IF, Trebak M (2010) A novel native store-operated calcium channel encoded by Orai3: selective requirement of Orai3 versus Orai1 in estrogen receptor-positive versus estrogen receptor-negative breast cancer cells. J Biol Chem 285:19173–19183. https://doi.org/10.1074/jbc.M110.102582
Faouzi M, Hague F, Potier M et al (2011) Down-regulation of Orai3 arrests cell-cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells. J Cell Physiol 226:542–551. https://doi.org/10.1002/jcp.22363
Dubois C, Vanden Abeele F, Lehen’kyi V et al (2014) Remodeling of channel-forming ORAI proteins determines an oncogenic switch in prostate cancer. Cancer Cell 26:19–32. https://doi.org/10.1016/j.ccr.2014.04.025
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dubois, C., Prevarskaya, N. (2018). Western Blotting and Co-immunoprecipitation of Endogenous STIM/ORAI and Protein Partners. In: Penna, A., Constantin, B. (eds) The CRAC Channel. Methods in Molecular Biology, vol 1843. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8704-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8704-7_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8702-3
Online ISBN: 978-1-4939-8704-7
eBook Packages: Springer Protocols