Human iPSCs were generated and differentiated as previously described (61 (link), 62 (link)). Briefly, primary fibroblasts were isolated from deidentified type I SMA patients and their unaffected relatives, infected with lentiviral constructs expressing OCT4, SOX2, NANOG, and LIN28 (61 (link)) or episomally expressed reprogramming plasmids (62 (link)) to generate iPSCs. Retinoic acid, sonic hedgehog, cAMP, ascorbic acid, BDNF, and glial cell line-derived neurotrophic factor (GDNF) were used to induce iPSC spheres to differentiate into motor neurons. Primary neurons from mouse spinal cords were cultured in Neurobasal (Life Technologies) supplemented with B27 (Life Technologies) as described (22 (link), 56 (link)). Briefly, spinal cords from E12.5 mouse embryos were dissected out and dissociated with 0.25% trypsin. After enriching motor neurons with OptiPrep density gradient centrifugation and BSA cushion, cells were seeded on glass coverslips coated with 20 μg/mL Poly-L-Lysine (PLL) (Sigma) and 8 μg/mL Laminin (Sigma) and grown in the presence of 50 μg/mL BDNF, 50 μg/mL CNTF and 25 μg/mL GDNF (PeproTech). Cell death was detected by the in situ cell death detection TMR red TUNEL kit. Cdk5 inhibitor BML259 (300 nM) (Cayman Chemical) and calpain inhibitor Calpeptin (10 μM) (Tocris Bioscience) were applied from weeks 3 to 6 into iPSC motor neuron medium to inhibit Cdk5 signaling.
Calpeptin
Manufactured by Bio-Techne
Sourced in United States
Calpeptin is a laboratory reagent produced by Bio-Techne. It functions as a selective and potent inhibitor of the calcium-activated protease calpain.
Automatically generated - may contain errors
Lab products found in correlation
Poly l lysine (pll), by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Laminin, by Merck Group (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Emetine, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Mg132, by Bio-Techne (1 mentions)
Puromycin, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Click it edu alexa fluor 488 hcs assay, by Thermo Fisher Scientific (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
Desmoplakin, by Santa Cruz Biotechnology (1 mentions)
Desmoglein, by Santa Cruz Biotechnology (1 mentions)
Total caveolin 1, by BD (1 mentions)
Ptp1b, by Santa Cruz Biotechnology (1 mentions)
Total src, by Cell Signaling Technology (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Crosslink magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
β catenin, by BD (1 mentions)
8 protocols using calpeptin
1
Generating iPSC-Derived Motor Neurons for SMA
2
Inhibitors of Protein Synthesis
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Cycloheximide, Emetine, Puromycin and Chloroquine were purchased from Sigma Chemical Co. (St. Louis, MO). Calpeptin and MG132 were purchase from Tocris Bioscience (Minneapolis, MN).
3
Calpeptin Modulates Neural Stem Cell Proliferation
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NSC were isolated from the SVZ of P0-3 Cast+/+ and Cast−/− mice and maintained in culture, as previously described (Morte et al., 2013 (link)). Dissociated NSC (n = 3) were plated on coverslips coated with 0.1 mg/ml poly-L-lysine (Sigma Aldrich, St Louis, MO, USA) until 60–70% confluency was reached, and then treated with calpeptin 25 μM (Tocris Bioscience, Bristol, UK) for 6 h (untreated cells were used as controls). The cells were kept with EdU 10 μM for the last 4 h before fixation with 4% paraformaldehyde/4% sucrose. EdU incorporation was processed using a commercially available kit (Click-iT®EdU Alexa Fluor®488 HCS Assay, Invitrogen, Paisley, UK) and nuclei were stained with 1 μg/ml Hoechst 33342, for 5 min. EdU-positive cells were counted in images acquired in an Axio Imager Z2 microscope (Zeiss, Jena, Germany).
4
Regulation of Caveolin-1 and β-Catenin
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All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. NS1643 was from Alamo Laboratories Inc. (San Antonio, TX, USA). Calpeptin was purchased from Tocris Bioscience (Minneapolis, MN, USA). PTP1B inhibitor was from Cayman Chemical (Ann Arbor, MI, USA). Phospho-Y14, total Caveolin-1, β-catenin, and caspase-3 antibodies were from BD Biosciences (San Jose, CA, USA). Phospho-Y416, Y527, and total Src were from Cell Signaling Technology (Danvers, MA, USA). PTP1B, desmoplakin, desmoglein, and plakophilin monoclonal Abs, normal mouse IgG, and protein A/G agarose beads were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). DAPI and all fluorescently labeled secondary antibodies were purchased from Molecular Probes (ThermoFisher Scientific, Waltham, MA, USA). Active β-catenin monoclonal antibody was purchased from MilliporeSigma (Burlington, MA, USA). HRP-conjugated goat-anti-mouse and goat-anti-rabbit secondary antibodies were from KPL (Gaithersburg, MD, USA). Lipofectamine 2000, ECL Super Signal kit, and Crosslink magnetic IP/Co-IP kit were from ThermoFisher Scientific (Waltham, MA, USA).
5
Cloning and Tagging of Human FAPP1 Protein
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Human FAPP1 cDNA was PCR amplified from the HeLa cell total mRNA and cloned into pcDNA-6.2/N-EmGFP-DEST (for N-terminal tagging) by using Gateway (Invitrogen) cloning technology as described previously (58 (link)). Ii-Str_SBP-EGFP-E-cadherin and Str-KDEL_ManII-SBP-EGFP were purchased from Addgene (plasmids 65288 and 65252). Polyethylenimine (Polysciences) and Lipofectamine 3000 (Invitrogen) were used for transfection. Z-VAD-FMK was purchased from Promega and was used at 50 μM. Cytochalasin D (cytD) was purchased from Tocris Bioscience and used at 5 μg/ml. Z-VAD-FMK or cytD was added 1 h before infection. Calpeptin was purchased from Tocris Bioscience and was used at 20 or 50 μM. Antibodies used for Western blot analysis were anti-E-cadherin (1:1,000) (24E10, 3195; Cell Signaling Technology), anti-β-actin (1:1,000) (13E5, 4970; Cell Signaling Technology), anti-β-catenin (1:100, ab16051; Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000, H12, sc-166574).
6
Inhibitor Preparation for Cellular Studies
7
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.
8
Neuronal Culture Treatments for Alzheimer's
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Glutamate (50 μM; Sigma), calpeptin (50 μM; Tocris Bioscience, Minneapolis, MN, USA), FCCP (0.25–2 μM; Sigma), rotenone (10–100 nM; Sigma), and okadaic acid (5–50 nM; Cayman Chemical, Ann Arbor, MI, USA) were added to neuronal cultures at the indicated final concentrations. Aβ oligomers were prepared as previously described (Song et al., 2014 ). Briefly, lyophilized Aβ peptides were dissolved in dimethyl sulfoxide, diluted in neurobasal without phenol red (Gibco‐BRL) to a final concentration of 1 μM, and incubated at 4°C for 16 hr. Prepared Aβ oligomers were added to neuronal cultures for the indicated times.
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