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Calpeptin

Manufactured by Merck Group
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Sourced in United States, Germany

Calpeptin is a laboratory reagent produced by Merck Group. It functions as a potent and selective inhibitor of calpains, a family of calcium-dependent cysteine proteases. Calpains play a role in various cellular processes. Calpeptin is commonly used in research applications to study the involvement of calpains in biological systems.

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Lab products found in correlation

Mg132, by Merck Group (5 mentions) Bapta am, by Merck Group (4 mentions) Penicillin, by Thermo Fisher Scientific (4 mentions) Streptomycin, by Thermo Fisher Scientific (4 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions) Pepstatin a, by Merck Group (3 mentions) Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Trypsin edta, by Thermo Fisher Scientific (3 mentions) Ionomycin, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Cycloheximide (chx), by Merck Group (2 mentions) Caspase 3, by Cell Signaling Technology (2 mentions) Z vad fmk, by Merck Group (2 mentions) Bafilomycin, by Merck Group (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) 3 methyladenine, by Merck Group (2 mentions) Y 27632, by Merck Group (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Bafilomycin a1, by Merck Group (2 mentions)

39 protocols using calpeptin

1

Synaptic Protein Expression Analysis

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Manganese (II) chloride tetrahydrate (MnCl2·4H2O), Calpains, Calpeptin, SynaptoGreenTMC4 (FM1-43) and L-glutamine were purchased from Sigma (Saint Louis, MO, USA). PrimeScript®RT Enzyme Mix I and SYBR®Premix Ex TaqTMII kits were from TaKaRa Biotech. Co. Ltd. Mouse β-actin primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit Syntaxin 1, VAMP-2 primary antibody were purchased from Abcam Ltd. (Hong Kong), mouse SNAP-25 N-terminal monoclonal and C-terminal polyclonal antibodies were purchased from Synaptic Systems (Germany). Horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibody and HRP conjugated anti-mouse secondary antibody were purchased from Abcam. Additional chemicals of analytical grade were obtained from local chemical suppliers.
Wang C., Xu B., Ma Z., Liu C., Deng Y., Liu W, & Xu Z.F. (2017). Inhibition of Calpains Protects Mn-Induced Neurotransmitter release disorders in Synaptosomes from Mice: Involvement of SNARE Complex and Synaptic Vesicle Fusion. Scientific Reports, 7, 3701.
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2

Mechanical Wounding and Cell Survival Assay

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MLO-Y4, OCY454, and primary osteocytes were wounded by glass beads, as previously described (8 (link), 19 (link)), to facilitate wounding large numbers of cells. Vitamin E (Sigma T3251, 220 μM) (19 (link)) or calpeptin (Sigma C8999, 20 μM) (20 (link)) were introduced as indicated 24 hours prior to wounding. Whole cell lysates were collected for western blotting as previously described (21 ), using antibodies against c-fos (Santa Cruz) and β-actin (Sigma). Please see Supplementary Methods for additional details.
To assess cell survival after mechanical wounding, MLO-Y4 cells were wounded by glass beads in the presence of lysine-fixable fluorescein-conjugated dextran (10 kDa, 5 mg/mL+10 mg/mL BSA) containing either 1.8 mM Ca2+, 1.8 mM Ca2++20 μM calpeptin, or 1.5 mM EGTA. Five minutes after wounding, cells were stained with propidium iodide (0.3 μg/mL) to detect dead cells (i.e., unrepaired PMD) and imaged on a multi-photon confocal microscope (Zeiss). Please see Supplementary Methods for additional details. The percentages of PMD-affected cells and dead cells were quantified (Bioquant).
Yu K., Sellman D.P., Bahraini A., Hagan M.L., Elsherbini A., Vanpelt K.T., Marshall P.L., Hamrick M.W., McNeil A., McNeil P.L, & McGee-Lawrence M.E. (2017). Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 36(2), 653-662.
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3

Preparation of Calcium Lactate and Calpeptin

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Lactate calcium salt (CaLac) and calpeptin were purchased from Sigma-Aldrich (St. Louis, MO, United States) and dissolved in distilled water. The solutions were stored at 4 °C until use.
Jeong K.Y., Sim J.J., Park M, & Kim H.M. (2022). Accumulation of poly (adenosine diphosphate-ribose) by sustained supply of calcium inducing mitochondrial stress in pancreatic cancer cells. World Journal of Gastroenterology, 28(27), 3422-3434.
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4

Protease Inhibitor Cocktail for In Vitro and In Vivo Studies

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4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochlorine (AEBSF, A8456), aprotinin (A1153), bestatin (Sigma B8385), pepstatin A (P5318), N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E64, E3132), leupeptin (L2884), N-ethylmaleimide (NEM, E3876), calpeptin (C8999), calpain inhibitor-I (A6185), -II (A6060), -III (M6690), pan-caspase inhibitor (V116), BAPTA-AM (A1076), ionomycin (I0634) were supplied by Sigma. Calpistatin peptide (208902), negative control peptide (208904), elastase inhibitor IV (324759) and cathepsin G inhibitor I (219372) were supplied by Calbiochem. Tryptase inhibitor nafamostat mesylate (NM, 3081) was supplied by Tocris. AEBSF, aprotinin, bestatin, pepstatin A, E64, leupeptin and NEM were used in vitro at concentrations according to manufacturer’s instructions (Sigma Inhib1). calpeptin, calpain, NE, CG, caspase and tryptase inhibitors, BAPTA-AM were used at 100 μM during in vitro studies. Calpistatin and control peptide were used at 10 μM during in vitro studies. Doses of inhibitors used in vivo and details of all other reagents used are described in other method sections.
Scott I.C., Majithiya J.B., Sanden C., Thornton P., Sanders P.N., Moore T., Guscott M., Corkill D.J., Erjefält J.S, & Cohen E.S. (2018). Interleukin-33 is activated by allergen- and necrosis-associated proteolytic activities to regulate its alarmin activity during epithelial damage. Scientific Reports, 8, 3363.
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5

Neurotransmitter Regulation of NGF Signaling

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Drugs were dissolved in HBSS, which was used as the experimental vehicle, and applied directly to the media containing NGF. Drugs concentrations are based on published literature: choline (Acros Organics, Geel, Belgium) (10 mM, 3 mM, and 1 mM) [22 (link)]; α-bungarotoxin (Thermofisher) (BGTX) (50 nM) [32 (link)]; calpeptin (Sigma Aldrich, St. Louis MO, USA)(26 μM) [33 ], ryanodine (Santa Cruz) (30 μM) [21 (link)]; Xestospongin C. (Tocris Biosciences, Bristol, UK.) (Xest C.) (1 μM) [20 (link)]; FK506 (Tocris Biosciences) (40 μM) [34 (link)]; Substance P (Tocris Biosciences) (Sub P) (1 μM) [20 (link)]; PNU 282987 (Tocris Biosciences) (10 μM) [35 (link)].
King J.R, & Kabbani N. (2018). Alpha 7 nicotinic receptors attenuate neurite development through calcium activation of calpain at the growth cone. PLoS ONE, 13(5), e0197247.
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6

Apoptosis Pathway Evaluation Protocol

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AITC, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM), calpeptin, 4′,6-diamidino-2-phenylindole (DAPI), N-acetylcysteine (NAC), propidium iodide (PI), and trypan blue were obtained from Sigma-Aldrich (Merck KGaA). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA), 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3 (link))], fetal bovine serum (FBS), 4-(6-acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-4′-methyl-2,2′(ethylenedioxy)dianiline-N, N,N,N′-tetraacetic acid tetrakis(acetoxymethyl) ester (fluo-3/AM), L-glutamine, Roswell Park Memorial Institute (RPMI)-1640 medium, penicillin, streptomycin, and trypsin-EDTA were obtained from Thermo Fisher Scientific, Inc. Caspase-3 and Caspase-9 Colorimetric Assay Kits were purchased from R&D Systems. Caspase-3 inhibitor Z-DEVD-FMK and caspase-9 inhibitor Z-LEHD-FMK were purchased from Merck Millipore. All primary antibodies were purchased from Cell Signaling Technology, Inc. or Santa Cruz Biotechnology, Inc. The goat anti-rabbit or anti-mouse immunoglobulin G (IgG) secondary antibodies conjugated with horseradish peroxidase (HRP) were also obtained from Cell Signaling Technology, Inc. Mitochondria/Cytosol Fractionation Kit was purchased from BioVision, Inc. The goat anti-mouse IgG-phycoerythrin (PE) secondary antibody was also obtained from Santa Cruz Biotechnology, Inc.
Chiang J.H., Tsai F.J., Hsu Y.M., Yin M.C., Chiu H.Y, & Yang J.S. (2020). Sensitivity of allyl isothiocyanate to induce apoptosis via ER stress and the mitochondrial pathway upon ROS production in colorectal adenocarcinoma cells. Oncology Reports, 44(4), 1415-1424.
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7

Podocyte Stress Response Mechanisms

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The conditionally immortalized human podocyte cell line was kindly provided by Dr. Moin A. Saleem (Academic Renal Unit, University of Bristol, UK). Podocytes were cultured in the medium which contained RPMI 1640 medium (Gibco, United States) with 10% fetal bovine serum (Gibco, United States), and insulin-transferrin-selenium (ITS) (Gibco, United States) at 33°C. Podocytes differentiated when cultured at 37°C for 10–14 days. The differentiated cells were treated with normal glucose (Control), 30 mM mannitol (HM) or 30 mM glucose (HG). Podocytes were respectively treated with 2-aminoethoxydiphenyl borate (2-APB, 100 μM, Sigma, United States), BAPTA-AM (10 μM, MedChemExpress, United States), roscovitine (10 μM, Sigma, United States), calpeptin (1 μM, Sigma, United States), thapsigargin (TG, 1 μM, Sigma, United States), 1-oleoyl-2-acetyl-glycerol (OAG, 100 μM, Sigma, United States) and DMSO. The X-treme GENE siRNA Transfection Reagent (Roche, Switzerland) was used to transiently transfect podocytes with TRPC6 siRNA, CDK5 siRNA, calpain-1 siRNA, or scrambled siRNA (Santa Cruz Biotechnology, United States) according to the manufacturer’s protocols. Transfected podocytes were examined within 24–48 h post-transfection.
Yu H., Chen Y., Ma H., Wang Z., Zhang R, & Jiao J. (2022). TRPC6 mediates high glucose-induced mitochondrial fission through activation of CDK5 in cultured human podocytes. Frontiers in Physiology, 13, 984760.
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8

Rotenone-Induced Neurodegeneration: Calpeptin Neuroprotection

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Rats were divided into four treatment groups: (1) control+ vehicle, (2) calpeptin, (3) Rotenone, and (4) Rotenone plus calpeptin. Rotenone (Sigma, St. Louis, MO, USA) was injected subcutaneously (s.c.) at a dose of 2 mg/kg body weight. Groups 3 and 4 received Rotenone s.c. daily for four days, and then every other day for 6 days. Group 4 also received calpeptin (Sigma, St. Louis, MO, USA) intraperitoneally (i.p.) daily at a dose of 25 µg/kg body weight. The calpeptin injections started one day after the 1st injection of Rotenone (nine injections total) and were injected 1 h after Rotenone injection.
Stock solution of Rotenone was prepared in dimethylsulfoxide (DMSO, Sigma). The working emulsified dilution was made in sunflower oil. The dose was prepared fresh every other day and stored in amber-colored glass vials. The calpeptin stock solution was prepared in DMSO, and working dilution was prepared in sterile saline. The working dilution was prepared fresh each time before use. The animal’s body weight was measured before starting the treatments and at the end of the treatments.
Zaman V., Drasites K.P., Myatich A., Shams R., Shields D.C., Matzelle D., Haque A, & Banik N.L. (2022). Inhibition of Calpain Attenuates Degeneration of Substantia Nigra Neurons in the Rotenone Rat Model of Parkinson’s Disease. International Journal of Molecular Sciences, 23(22), 13849.
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9

Pharmacological Modulation of Motor Neurons

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Motor neurons were treated with various drugs. The drugs used in this study are TTX (T8024, Sigma), riluzole (R116, Sigma), 3-methyladenine (3-MA, M9281, Sigma), MG132 (M7449, Sigma), nutlin3 (N6287, Sigma), calpeptin (C8999, Sigma) and bafilomycin A (B1793, Sigma), N-methyl D-aspartate (NMDA, Cay-14581, Biomol), rapamycin (#9904, Cell Signaling), Torin-1 (#CALB475991, Calbiochem), 10-NCP (#2338, Biovision) and cycloheximide (#01810, Sigma). Concentrations of drugs used in this study can be found in the figure legends.
Gonçalves I.D., Brecht J., Thelen M.P., Rehorst W.A., Peters M., Lee H.J., Motameny S., Torres-Benito L., Ebrahimi-Fakhari D., Kononenko N.L., Altmüller J., Vilchez D., Sahin M., Wirth B, & Kye M.J. (2018). Neuronal activity regulates DROSHA via autophagy in spinal muscular atrophy. Scientific Reports, 8, 7907.
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10

Compound Procurement for Cell Assays

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Camptothecin, capsaicin, BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) and calpeptin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All cell culture reagents, including RPMI-1640, FBS, Trypsin-EDTA, and HEPES, were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Sodium pyruvate, glucose, and penicillin-streptomycin solutions were obtained from Corning (NY, USA).
Friedman J.R., Perry H.E., Brown K.C., Gao Y., Lin J., Stevenson C.D., Hurley J.D., Nolan N.A., Akers A.T., Chen Y.C., Denning K.L., Brown L.G, & Dasgupta P. (2017). Capsaicin Synergizes with Camptothecin to Induce Increased Apoptosis in Human Small Cell Lung Cancers via the Calpain Pathway. Biochemical pharmacology, 129, 54-66.
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