All chemicals were purchased from Fisher Scientific unless otherwise specified. β1-AR was from Santa Cruz Biotechnologies (Dallas, TX, U.S.A.). Cx43, PKA, P-PKA (T197), ERK1/2, P-ERK1/2 (T202/Y204), P-CamKII (T286), CamKII, calreticulin, calmodulin, P-S6 (S240/244), S6, and HRP-conjugated mouse and rabbit secondary antibodies were from Cell Signaling Technology (Boston, MA, U.S.A.). HCN2 and Kv4.2 antibodies were from Neuro-Mab (Davis, CA, U.S.A.). NCX-1 was from Alomone Labs (Jerusalem, Israel).
Calmodulin
Manufactured by Cell Signaling Technology
Sourced in United States
Calmodulin is a calcium-binding protein that plays a crucial role in various cellular processes. It acts as a calcium sensor and can bind to and regulate the activity of numerous target proteins, enabling them to respond to changes in intracellular calcium levels. Calmodulin is a highly conserved and ubiquitous protein found in all eukaryotic cells.
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Lab products found in correlation
β actin, by Cell Signaling Technology (2 mentions)
Calreticulin, by Cell Signaling Technology (1 mentions)
P erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
β1 ar, by Santa Cruz Biotechnology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P erk, by Affinity Biosciences (1 mentions)
C raf, by Affinity Biosciences (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
Caspase 8, by Merck Group (1 mentions)
Caspase 3 and, by Abcam (1 mentions)
Calpain 1, by Abcam (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Tradd, by Cell Signaling Technology (1 mentions)
Tnf α, by Cell Signaling Technology (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
α synuclein, by Cell Signaling Technology (1 mentions)
6 protocols using calmodulin
1
Cardiac Signaling Protein Assay
2
Western Blot Analysis of EMT Markers
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The detailed experimental procedures were as described previously 20 (link). Briefly, RIPA lysis buffer was used to extract proteins from cell samples and tissues. Then we separated equal amounts of lysate protein by SDS-PAGE, and then transferred them onto a PVDF membrane. The following primary antibodies were used: HRC (Sigma-Aldrich, SAB1303636), c-Raf (Affinity Biosciences, Cincinnati, OH, USA; AF6065), phosphorylated (p)-c-Raf (Affinity Biosciences, AF3065), MEK (Affinity Biosciences, AF6385), p-MEK (Affinity Biosciences, AF8035), ERK (Affinity Biosciences, AF0155), p-ERK (Affinity Biosciences, AF1015), N-cadherin (Cell Signaling Technology, Danvers, MA, USA; #13116S), Calmodulin (Cell Signaling Technology, #S35944), Vimentin (Cell Signaling Technology, #5741S), E-cadherin (Cell Signaling Technology, #3195S), Snail (Cell Signaling Technology, #S3879), Slug (Cell Signaling Technology, #9585S), and GAPDH (Abcam, Cambridge, MA, USA; ab9485).
3
Western Blot Analysis of Cellular Signaling
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Cells were lysed by RIPA buffer (50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 1% (v/v) NP-40; 5 mmol/L EDTA; 1 mmol/L DTT; 0.5% sodium deoxycholate; 0.1% SDS; 1 mmol/L Na3VO4; and 1 mmol/L PMSF) and centrifuged at 12,000×g for 30 min at 4°C. Supernatant proteins were determined by Bradford method using BSA as standard and measured at 595 nm. After SDS page separation, proteins were transferred onto a PVDF membrane and revealed by ECL detection reagents. Primary antibodies used in the study included TNF-alpha, TRADD, β-actin, CaM, cytochrome C, calmodulin from Cell signaling technology (Denvers, MA, USA); caspase 8 were from Millipore (Billerica, MA, USA); RIP3 and calpain1 were from Biovision (Milpitas, CA, USA); caspase 3 and were from Abcam (Cambridge, UK).
4
Neuroblastoma Cell Culture and Analysis
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Human neuroblastoma cells line SK-N-SH (ATCC® HTB11™) were purchased from American Type Culture Collection (ATCC, USA). Minimum essential medium (MEM), fetal bovine serum (FBS), penicillin-streptomycin, Trypsin, and phosphate buffered saline (PBS) solutions were obtained from Gibco Life Technologies (Invitrogen, USA). Dimethyl sulfoxides (DMSO), retinoic acid (RA), thiazolyl blue tetrazolium bromide (MTT), and forskolin were purchased from Sigma-Aldrich (USA). Morphine sulphate pentahydrate (M-35-SU) and d,I-methadone.HCl (MET-637) were purchased from Lipomed AG (Switzerland). Isobutylmethylxanthine (IBMX) and radioimmunoprecipitation assay (RIPA) buffer and protease inhibitor were purchased from Amresco, USA. The antibodies used, α-synuclein, calmodulin, synaptotagmin 1, VAMP 2, anti-β-actin, and horseradish peroxidase (HRP) were purchased from Cell Signaling Technology (Massachusetts). WesternBright™ ECL and WesternBright Peroxide were purchased from Advansta (USA).
5
Endoplasmic Reticulum Stress Response Pathway Analysis
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RPMI-1640, fetal bovine serum (FBS), antibiotic, antimycotic, bovine serum albumin (BSA), Ca2+ ionophore, EGTA, Thapsigargin (Tg), and Staurosporine (St) were purchased from Sigma Aldrich, USA. Anti-phospho-PERK (Thr980), PERK, phospho-eIF2α (S51), eIF2α, TRAF, IRE1α, BiP, Grp94, PDI, CHOP, Ero1-Lα, phospho-PKC βII (S660), phospho-PKC α/β (T638/641), Pan PKC, Calmodulin, NFAT-3, Calnexin, Caspase 12 and β-actin antibodies were purchased from Cell Signaling Technology, USA. NE-PER™ nuclear and cytoplasmic extraction reagents were from Pierce, Thermo-scientific, USA. ER-Tracker™ dyes for live-cell endoplasmic reticulum labeling was from Molecular Probes, USA. Biotinylated Sambucus nigra agglutinin (SNA), Maackia amurensisagglutinin (MAA), Peanut Agglutinin (PNA), Ricinus Communis Agglutinin (RCA) and Erythrina Cristagalli Lectin (ETC) from Vector LaboratoriesCA, US. Ca2+ Ionophore, EGTA, and N-acetyl-L-cysteine (NAC) were purchased From Sigma Aldrich
Mahanine was purified from fresh leaves of a native Indian plant, Murraya koenigii belonging to the family Rutaceae. The purity was confirmed by HPLC. LC-MS, [1H] and [13C] NMR spectral data analysis established its structure as mahanine51 .
Mahanine was purified from fresh leaves of a native Indian plant, Murraya koenigii belonging to the family Rutaceae. The purity was confirmed by HPLC. LC-MS, [1H] and [13C] NMR spectral data analysis established its structure as mahanine51 .
6
Daphnetin Modulates T Cell Responses
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Daphnetin (purity>98%, catalog number:110900) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Daphnetin was diluted in culture medium with 0.1% dimethylsulfoxide (DMSO, Sigma, St. Louis, MO, USA) and then sterile-filtered before used. All controls and samples contained the same concentrations of DMSO.
Concanavalin A (ConA) and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The mAbs against mouse CD3, CD4, CD8, IFN-γ, IL-2, IL-4 and IL-6 ELISA kits were purchased from Biolegend (California, USA). RPMI-1640 medium was purchased from HyClone (Logan, UT), and fetal bovine serum (FBS) was obtained from Invitrogen-Gibco (Grand Island, NY). Primary antibodies used for Western analysis Calmodulin, Calcineurin (CaN), Ca2+/Calmodulin-dependent kinase II (CaMKII), P-CaMKII, NF-κB, which were purchased from Cell Signaling (beverly, MA), NFAT2 antibodies was purchased from Abcam (Cambridge, MA), β-actin was obtained from Tianjin Sungene Biotech Co., Ltd. Goat anti-mouse IgG, Goat anti-mouse FITC, Peroxidase-conjugated AffiniPure Goat anti-mouse IgG (H + L) were purchased from Protein Tech Group, 2, 4-Dinitrofluorobenzene (DNFB) and cyclophosphamide (CTX) were obtained from Sigma-Aldrich (St. Louis, MO).
Concanavalin A (ConA) and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The mAbs against mouse CD3, CD4, CD8, IFN-γ, IL-2, IL-4 and IL-6 ELISA kits were purchased from Biolegend (California, USA). RPMI-1640 medium was purchased from HyClone (Logan, UT), and fetal bovine serum (FBS) was obtained from Invitrogen-Gibco (Grand Island, NY). Primary antibodies used for Western analysis Calmodulin, Calcineurin (CaN), Ca2+/Calmodulin-dependent kinase II (CaMKII), P-CaMKII, NF-κB, which were purchased from Cell Signaling (beverly, MA), NFAT2 antibodies was purchased from Abcam (Cambridge, MA), β-actin was obtained from Tianjin Sungene Biotech Co., Ltd. Goat anti-mouse IgG, Goat anti-mouse FITC, Peroxidase-conjugated AffiniPure Goat anti-mouse IgG (H + L) were purchased from Protein Tech Group, 2, 4-Dinitrofluorobenzene (DNFB) and cyclophosphamide (CTX) were obtained from Sigma-Aldrich (St. Louis, MO).
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