Drug hits that inhibited at least 66% of LF FRET reaction were also tested for their ability to inhibit FRET reactions of human furin (New England Biolabs), calpain 1 (BioVision), cathepsin B (EMD Millipore), and caspases-1 and -3 (BioVision). The fluorescently labelled substrate peptide for furin FRET assay was purchased from Peptides International. The positive control furin Inhibitor I was purchased from EMD Millipore. The calpain Inhibitor 1, ALLN, was purchased from BioVision and used as a control. Amodiaquine (Sigma-Aldrich) was used as a control inhibitor of cathepsin B. The universal caspase inhibitor Z-VAD-FMK (BioVision) was used as a control. Rates of reactions were quantified by the Microsoft Excel LINEST function.
Calpain 1
Manufactured by Abcam
Sourced in United States
Calpain-1 is a calcium-dependent intracellular cysteine protease. It plays a role in cellular processes such as signal transduction, cell motility, and apoptosis. Calpain-1 is involved in the cleavage of various cellular substrates.
Automatically generated - may contain errors
Lab products found in correlation
Calpain 2, by Abcam (3 mentions)
Calpastatin, by Abcam (2 mentions)
Timp2, by Boster Bio (2 mentions)
Mt1mmp, by Boster Bio (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Z vad fmk, by Abcam (1 mentions)
Amodiaquine, by Merck Group (1 mentions)
Cathepsin b, by Merck Group (1 mentions)
Calmodulin, by Cell Signaling Technology (1 mentions)
Caspase 8, by Merck Group (1 mentions)
Caspase 3 and, 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)
β actin, by Cell Signaling Technology (1 mentions)
Calpeptin, by Bio-Techne (1 mentions)
Anti gst, by Santa Cruz Biotechnology (1 mentions)
Tgf β1, by Abcam (1 mentions)
Hif 1α, by Abcam (1 mentions)
11 protocols using calpain 1
1
Inhibition of Proteolytic Enzymes
2
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).
3
In Vitro Cleavage of Recombinant GST-DLP1 by Calpain
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In vitro cleavage of recombinant GST‐DLP1 protein by calpain was performed as previously described (Garg, Timm, Mandelkow, Mandelkow, & Wang, 2011 ). Briefly, recombinant GST‐DLP1 (160 ng; Abnova, Walnut, CA, USA) was incubated with calpain‐1 (Biovision, San Francisco, CA, USA) in reaction buffer for various times with or without calpeptin (50 μM; Tocris Bioscience, Minneapolis, MN, USA). After being incubated for the indicated times, the reaction mixture was mixed with an equal volume of 2× SDS sample buffer and boiled for 10 min. Samples were subjected to SDS‐PAGE followed by Western Blotting with anti‐DLP1 D6C7 (Cell Signaling Technology, Danvers, MA, USA) or anti‐GST (Santa Cruz, Dallas, TX, USA) antibodies.
4
Protein Expression Analysis in VSMCs
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VSMCs were lysed in RIPA buffer. Protein concentrations were quantified with protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Bio-Rad). The membranes were incubated overnight with primary antibodies against HIF-1α (1:1000, Abcam), calpastatin, TGF-β1 (1:2000, Abcam), calpain-1, calpain-2, a-II spectrin (1:3000, Abcam), MMP2, TIMP2 (1:400, Boster), MT1MMP (1:200, Boster), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, Cell Signaling Technology). At room temperature, membranes were incubated with corresponding secondary antibodies for 1 hour. Quantitative analysis was performed with Image J software (NIH). All samples were run in triplicate and averaged.
5
Honey Bee Protein Level Analysis
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The honey bees used for protein level analysis were divided into four groups: no treatment (con.), sodium butyrate only (NaB), DWV only (DWV) and NaB+DWV (N/D). Sodium butyrate (10 mM) in ddH2O was used in the feeding assay for 7 days. Protein was extracted from the brains of the honey bees for Western blot analysis using a kit from Millipore (Hu et al., 2017 (link)). The samples were subjected to electrophoresis on SDS-PAGE gels with equal amounts of loaded samples. The proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell) by electroblotting for 1 h. Membranes were then blocked with PBS containing 5% skimmed milk and 0.05% Tween-20 and incubated with primary anti-mouse antibodies against Acetyl-H3 (1:5000 dilution) (Millipore), Acetyl-H4 (1:5000 dilution) (Millipore), Calpain-1 (1:5000 dilution) (ABcam) and Actin (1:3000 dilution) (Millipore), followed by secondary rabbit anti-mouse antibody conjugated with horseradish peroxidase (Millipore). The proteins were detected with an enhanced chemiluminescence system (Immobilon Western, Millipore) (Hu et al., 2018 (link)). Each group included three biological replicates.
6
Analyzing Cortical Injury Markers
7
Western Blot Analysis of Cell Signaling
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For western blot analysis, cells were lysed with ice-cold RIPA buffer supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Roche Diagnostics). Lysates were clarified by centrifugation at 13000 rpm for 30 min, after which the supernatants were harvested. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo). Equal amounts of protein were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). After blocking nonspecific binding sites on the membranes with 5% skim milk or 5% BSA for 2 hours at RT, the membranes were incubated with the indicated primary antibodies overnight at 4°C and then with the appropriate secondary antibodies for 1 hour at RT. Antibodies against Cyclin B, Cyclin D1, E2F1, survivin, PARP, EGFR, FAK, Src, α-tubulin, GAPDH (Cell Signaling Technology), TfR1, calpain-1, calpain-2 (Abcam), c-Met (Invitrogen), and c-MYC (Santa Cruz Biotechnology) were obtained commercially. Immunoreactive bands were visualized with HRP-conjugated secondary antibodies and a chemiluminescent substrate by exposure to X-ray film.
8
Carotid Artery Histological Analysis
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After perfusion with saline, carotid arteries were fixed with 4% paraformaldehyde and embedded in paraffin. Thin sections (4–5 μm) were stained with haematoxylin & eosin (HE). The areas of intima and media of the carotid artery were measured using Image-Pro Plus software. Cell proliferation was assessed by immunohistochemical staining of proliferation cell nuclear antigen (PCNA) with the corresponding antibody (1:200, Abcam). Protein expression was evaluated by immunohistochemical staining using antibodies against calpastatin, calpain-1, calpain-2 (1:100, Abcam), MMP2, MT1MMP, TIMP2, and collagen I (1:100, Boster).
9
Calpain-Mediated Desmin Proteolysis
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Retinal lysates (10 μg total protein per retina) or 200 ng of Desmin-OE lysate in RIPA buffer, omitting protease inhibitor cocktail, were diluted 1:5 (v/v) in calpain digest buffer: Tris-HCl pH 7.5, 150 mM NaCl, 2 mM CaCl2, 2.5 mM β-mercaptoethanol, 0.2% (v/v) Triton X-100 and 0.3% (v/v). Parallel reactions were set up with or without protease inhibitor EGTA (at 2 mM) combined with 2 U of active Calpain-1 (Abcam, #ab91019), and subjected to rapid digest for 1 min or 5 min at 30 °C before quenching in 1:1 v/v 2x Laemmli sample buffer before and boiling for 5 min prior to analysis by SDS-PAGE.
10
Western Blot Analysis of Calpain Signaling
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Protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Membranes were blocked in 5% skim milk for 1 h at 25°C and incubated with calpain 1 (1 : 1000, Abcam, Cambridge, MA, USA), calpain 2 (1 : 1000, Abcam), calpastatin (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), and p-GSK3β (Ser 9) (1 : 1000, Cell Signaling Technology) overnight at 4°C. The membranes were then washed with TBST three times and incubated for 1 h with appropriate HRP-conjugated secondary antibodies at room temperature. Blots were washed three times with TBST, and chemiluminescence (ECL) was used to detect the bands when exposed to an X-ray film (Tanon, ABclonal, Woburn, Massachusetts, USA).
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