Stim1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

STIM1 is a protein that functions as a calcium sensor in the endoplasmic reticulum (ER) membrane. It plays a key role in the activation of store-operated calcium entry (SOCE), a process that regulates calcium homeostasis in cells.

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Lab products found in correlation

Orai1, by Santa Cruz Biotechnology (5 mentions) β actin, by Merck Group (2 mentions) Orai2, by Santa Cruz Biotechnology (2 mentions) E cadherin, by Santa Cruz Biotechnology (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions) Anti mouse horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Trpc1, by Santa Cruz Biotechnology (1 mentions) Amaxa biosystem, by Lonza (1 mentions) Immobilon p, by Merck Group (1 mentions) Col2a1, by Santa Cruz Biotechnology (1 mentions) Runx2, by Cell Signaling Technology (1 mentions) Phospho akt, by Cell Signaling Technology (1 mentions) Phospho p38, by Cell Signaling Technology (1 mentions) Flag tag (flag), by Merck Group (1 mentions) Phospho erk, by ABclonal (1 mentions) Phospho jnk, by Cell Signaling Technology (1 mentions) β actin, by Proteintech (1 mentions)

Close spelling variants of this product

Stim1 antibody (2 citations)

9 protocols using stim1

Western Blot Analysis of Lung Proteins

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Total proteins from frozen left lung samples were isolated using a RIPA lysis buffer
(Thermo Fisher) supplemented with a Protease inhibitor tablet cocktail according to
manufacturer's instructions (Pierce™, Thermo Fisher). Protein concentrations were
calculated using DC Protein Assay (Bio-Rad Hercules, CA, USA) following the manufacture
instructions. Isolated proteins were stored at −80°C until use. Ten micrograms of protein
were resolved in an SDS-PAGE (10–12% gradient gels) followed by transferring of proteins
onto nitrocellulose membranes. After appropriate blocking (5% nonfat dried milk) the blots
were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076),
TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz,
sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry
milk overnight at 4°C. After washing, membranes were incubated with horseradish
peroxidase-conjugated secondary anti-mouse antibody (Jackson ImmunoResearch, West Grove,
PA, USA) diluted 1:5000 in 5% powdered nonfat dry milk. Levels of proteins were normalized
to the β-actin (Sigma-Aldrich, AC-74) content of the same sample. The signals obtained
were scanned with densitometry through a detection device by chemiluminescence (Odyssey
Imaging System LI-COR Biosciences, Lincoln, NE, USA) quantified with the Image J software
(NIH, Bethesda, MD, USA).

Castillo-Galán S., Arenas G.A., Reyes R.V., Krause B.J, & Iturriaga R. (2020). Stim-activated TRPC-ORAI channels in pulmonary hypertension induced by chronic intermittent hypoxia. Pulmonary Circulation, 10(1 Suppl), 13-22.

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Silencing Kv10.1, Orai1, SPCA2, and Stim1

Cited 1 time

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Transfection of cells was performed using nucleofection technology (Amaxa Biosystems, Lonza, Aubergenville, France) according to the protocol previously described^{11 (link)}. Cells were transiently transfected with siRNA directed against Kv10.1 (Dharmacon Research, Chicago, IL), Orai1 (Dharmacon Research, Chicago, IL), SPCA2 (Kaneka Eurogentec S.A.,Seraing, Belgium) and Stim1 (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), or with scrambled siRNA as a control (siCtl) (siGENOME Non-Targeting siRNA, Dharmacon Research, Chicago, IL), and used 72 h after transfection.

Peretti M., Badaoui M., Girault A., Van Gulick L., Mabille M.P., Tebbakha R., Sevestre H., Morjani H, & Ouadid-Ahidouch H. (2019). Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership. Scientific Reports, 9, 1175.

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Quantifying Epithelial-Mesenchymal Transition

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Extracts of total cellular proteins were prepared by scraping the cells into Mammalian Protein Extraction Reagent (Thermo Scientific Pierce, No. 78503, Shanghai, China). The protein samples were separated on the 12% SDS-PAGE and electrotransferred onto polyvinylidene difluoride-nitrocellulose membranes (Immobilon-P; Millipore Corporation, Bedford, MA, USA). The following primary antibodies were used: E-cadherin (SC-8426, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Cytokeratin19 (CK19) (SC-56371, Santa Cruz Biotechnology, Inc.), Zeb1 (ABIN755113, Antibodies-online, Shanghai, China), Snail1 (ABIN182776, Antibodies-online, Shanghai, China), Sox9 (SC-20095, Santa Cruz Biotechnology, Inc.), Col2A1 (SC-52658, Santa Cruz Biotechnology, Inc.), Runx2 (No. 12556, Cell Signaling Technology, Boston, MA, USA), OPN (SC-21742, Santa Cruz Biotechnology, Inc.), STIM1 (SC-166541, Santa Cruz Biotechnology, Inc.), ORAI1 (SC-68895, Santa Cruz Biotechnology, Inc.). HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Boster, BA1051, Wuhan, China) were used and detected by electrochemiluminescence (Amersham Pharmacia Biotech, Aylesbury, UK).

He D., Lu Y., Hu H., Zhang J., Qin B., Wang Y., Xing S., Xi Q, & Wang S. (2015). The Wnt11 Signaling Pathway in Potential Cellular EMT and Osteochondral Differentiation Progression in Nephrolithiasis Formation. International Journal of Molecular Sciences, 16(7), 16313-16329.

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Protein Expression Analysis Protocol

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Briefly, polyacrylamide gel electrophoresis was used to separate proteins which extracted from cells, and transferred onto polyvinylidene fluoride membranes. Membranes were incubated with primary antibodies including: FLAG (1:2000; Sigma), E-cadherin (1:500; Santa Cruz, Dallas, TX, USA), N-cadherin (1:500; Santa Cruz), Vimentin (1:1000; Cell Signalling Technology, Beverly, MA, USA), Snail (1:1000; Cell Signalling Technology), JNK (1:1000; Cell Signalling Technology), phospho-JNK (1:500; Cell Signalling Technology), p38 (1:1000; Cell Signalling Technology), phospho-p38 (1:500; Cell Signalling Technology), AKT (1:1000; Cell Signalling Technology), phospho-AKT (1:500; Cell Signalling Technology), FAK (1:1000; Cell Signalling Technology), phospho-FAK (1:500; Cell Signalling Technology), ERK (1:1000; ABclonal Biotech Co., Ltd., Cambridge, MA, USA), phospho-ERK (1:500; Abclonal), IP3R (1:1000; Santa Cruz), STIM1 (1:500; Santa Cruz), STIM2 (1:500; Santa Cruz), SERCA2 (1:500; Santa Cruz), ORAI1 (1:500; Santa Cruz), ORAI2 (1:500; Santa Cruz), ORAI3 (1:500; Santa Cruz), β-actin (1:3000; Proteintech) and then with HRP-conjugated secondary antibody. At last, an enhanced chemiluminescence assay was used to detect the reactions. All original western blots were provided as supplementary materials.

Song S., Liu B., Zeng X., Wu Y., Chen H., Wu H., Gu J., Gao X., Ruan Y, & Wang H. (2022). Reticulon 2 promotes gastric cancer metastasis via activating endoplasmic reticulum Ca2+ efflux-mediated ERK signalling. Cell Death & Disease, 13(4), 349.

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Cell Lysis and Protein Detection

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Cells were solubilized in triple detergent buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 mg/mL of phenylmethylsulfonyl fluoride) supplemented with phosphatase inhibitors (10 mM NaF, 10 mM b-glycerophosphate, 0.1 mM sodium vanadate) and protease inhibitor cocktail (Sigma) and briefly sonicated. Protein concentration was determined with the Bradford method (Bio-Rad Laboratories). The mouse monoclonal antibodies to ICP8, CD63, VP16, gD, CIN85 (Santa Cruz), β-actin, Flag epitope (Sigma), ARF6 (Thermo Fisher Scientific), STING (R&D systems), Us11 (kindly provided by Dr. Roizman-University of Chicago), and STIM1 (Santa Cruz) were used in a 1:1,000 dilution. The rabbit polyclonal phospho-STING (Ser-366), TBK1, and phospho-TBK1 (Ser-172) antibodies (Cell Signaling Technology) were used in a 1:500 dilution. The anifrolumab (Invitrogen) was used at 1 µg/mL. Proteins were visualized with 5-bromo-4-chloro-3 indolylphosphate-nitroblue tetrazolium) or with ECL western blotting detection reagents (Amersham Biosciences).

Dogrammatzis C., Saud R., Waisner H., Lasnier S., Suma S.M., Grieshaber B, & Kalamvoki M. (2024). Tracing the STING exocytosis pathway during herpes viruses infection. mBio, 15(4), e00373-24.

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Western Blot Analysis of Cell Lysates

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Standard denaturing SDS-PAGE techniques were applied to hASM cell lysates. Proteins were transferred onto PVDF membrane, blocked with 5% non-fat milk and probed with antibodies of interest. Primary antibodies raised in mouse or rabbit against TRPC3, ß-Actin, CD38, STIM1, Orai1, GAPDH, PCNA, and Cyclin E were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein detection was performed using LiCor IR-conjugated secondary anti-mouse or anti-rabbit antibodies, detected with a LiCor Odyssey gel documentation system.

Wylam M.E., Sathish V., VanOosten S.K., Freeman M., Burkholder D., Thompson M.A., Pabelick C.M, & Prakash Y.S. (2015). Mechanisms of Cigarette Smoke Effects on Human Airway Smooth Muscle. PLoS ONE, 10(6), e0128778.

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Immunoprecipitation and Calcium Signaling

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FUGENE transfection reagent was purchased from Promega (Madison, WI, USA). Primary antibodies against phospho-tyrosine (PY20, PY99, and PY350), Orai1, Stim1, GFP as well as A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-Pyk2 and pan Pyk2 antibodies were purchased from Cell Signaling Technologies (Beverly, MA, USA). Thapsigargin was purchased from EMD Millipore (MA, USA). Paraformaldehyde was purchased from Fisher Scientific (Hampton, NH, USA). Fura 2-AM was purchased from Life Technologies (Carlsbad, CA, USA). Scrambled and Pyk2 siRNA were purchased from Ambion (Foster City, CA). All details are described in Supplemental Experimental Procedures.

Yazbeck P., Tauseef M., Kruse K., Amin M.R., Sheikh R., Feske S., Komarova Y, & Mehta D. (2017). STIM1 Phosphorylation at Y361 Recruits Orai1 to STIM1 Puncta and Induces Ca2+ Entry. Scientific Reports, 7, 42758.

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Western Blot Analysis of ER Stress Markers

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Tissue specimens or cells were lysed using Western & IP Cell lysis Kit (Beyotime, Jiangsu, China) supplemented with protease inhibitor cocktail (Merck, CA, USA). The protein content was determined by BCA Protein Assay Kit (Beyotime). 40 μg of protein was separated by 8% SDS-PAGE and moved to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Blots were blocked and probed with antibodies against Stim1 (1:500), GAPDH (1:1000) (Santa Cruz Biotechnology), GRP78, CHOP, ATF4 (1:1000, Cell Signaling Technology Inc., Beverly, MA, USA) overnight at 4 °C. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and then visualized by ECL detection kit (Beyotime).

Sun X., Wei Q., Cheng J., Bian Y., Tian C., Hu Y, & Li H. (2017). Enhanced Stim1 expression is associated with acquired chemo-resistance of cisplatin in osteosarcoma cells. Human Cell, 30(3), 216-225.

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Antibody Characterization and Plasmid Identification

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Antibodies used in this study are as follows: alpha-Tubulin (Mouse, Sigma, T9026-100UL), GAPDH (Mouse, Invitrogen, MA5-151738), Calreticulin (Mouse, Abcam, ab22683), TAOK2, Rabbit, Sigma, HPA010650), Rtn3a (Rabbit, ProteinTech,12055-2-AP), Acetylated alpha Tubulin (Mouse, Sigma, T6793), GST (Mouse, Invitrogen), GM130 (Mouse, BD Labs, 610822), TAOK2-Cterm (Rabbit), Tom20 (Mouse, Santa Cruz Biotech, sc-17764), Stim1 (Mouse, Santa Cruz Biotech, sc-166840), GFP (Mouse, Roche, 11 814 460 001), Phospho-TAO2 (S181) (Rabbit, R&D Systems, PPS037). Addgene Plasmids used in this study are as follows: mCh-Sec61 beta (#49155), sfGFP-C1 (#54579), ER-mRFP (#62236), EGFP-Sec22b (#101918).

Nourbakhsh K., Ferreccio A., Bernard M.J., & Yadav S. (2021). TAOK2 is an ER-localized Kinase that Catalyzes the Dynamic Tethering of ER to Microtubules.

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