CNN and isomaltitol were produced by Hayashibara Co. Ltd (Okayama, Japan). D-(+)-mannose, D-(+)-glucosamine, hydrochloride theophylline, ammonium chloride (NH4Cl), and L-DOPA (3-(3,4-dihydroxyphenyl)-L-alanine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). LY294002 was obtained from Calbiochem (Darmstadt, Germany). α-melanocyte-stimulating hormone (α-MSH) was purchased from Sigma Aldrich (St. Louis, MO). Kojic acid (5-hydroxy -2-(hydroxymethyl)-4H-pyran-4-one) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Rabbit anti-tyrosinase, rabbit anti-TRP1, rabbit anti-TRP-2, and rat anti-LAMP-1 antibodies were purchased from Santa Cruz (Dallas, Texas). Rabbit anti-Pmel17(gp100) antibody was purchased from Abcam (Cambridge, UK). Mouse anti-Pmel17(HMB45) antibody and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Dako (Glostrup, Denmark). Mouse anti-MITF antibody was purchased from Exalpha Biologicals (Shirley, MA). Mouse anti-actin antibody was purchased from EMD Millipore (Temecula, CA). Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies were purchased from Life Technologies (Carlsbad, CA). Leupeptin and pepstatin were obtained from Peptide Institute, Inc. (Osaka, Japan). Complete EDTA-free Protease Inhibitor Cocktail was purchased from Roche Diagnostics (Basel, Switzerland).
Pepstatin
Manufactured by Peptide Institute
Sourced in Japan
Pepstatin is an inhibitor of aspartic proteases, such as pepsin, renin, and cathepsin D. It is a pentapeptide composed of the amino acids isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-L-alanine. Pepstatin is commonly used in research applications to study the role of aspartic proteases in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Leupeptin, by Peptide Institute (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
D mannose, by Fujifilm (1 mentions)
Ammonium chloride nh4cl, by Fujifilm (1 mentions)
α melanocyte stimulating hormone α msh, by Merck Group (1 mentions)
Rabbit anti trp1, by Santa Cruz Biotechnology (1 mentions)
Rat anti lamp 1, by Santa Cruz Biotechnology (1 mentions)
Mouse anti actin antibody, by Merck Group (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Nacalai Tesque (1 mentions)
4 protocols using pepstatin
1
Melanogenesis Regulation Pathway Analysis
2
Cathepsin Inhibitors for Protease Assays
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In addition to the new cathepsin V inhibitors identified in this study, the following well-established cathepsin inhibitors were used as controls: E-64 and its cell-permeable derivative E-64d (both Sigma-Aldrich) as general cysteine cathepsin inhibitors and CLIK-148, morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS), and pepstatin (Peptide Institute Inc, Osaka, Japan) [54] (link) for cathepsins L, S, and D, respectively. The inhibitor concentrations used in the assays were selected according to the literature for CLIK-148 [55] (link) and LHVS [56] (link) or used at the same concentrations as our new inhibitors. CLIK-148 and LHVS were kindly provided by prof. Nobuhiko Katunuma (University of Tokushima, Japan) and dr. James MeKerrow (University of California, San Francisco, USA), respectively.
3
Lysosomal Inhibitor Incubation Protocol
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MDMs or transfected 293 T cells were incubated with the lysosomal inhibitors (purchased from Peptide Institute, Osaka, japan), leupepetin (70 μM) and pepstatin (10 μM). Leupeptin and pepstatin was dissolved in H2O and DMSO, respectively, and the same volumes of H2O and DMSO were used as vehicle controls.
4
Quantifying Lung Cytokine Profiles
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Lavaged lungs (4-8 mice/group) were each homogenized in 3 ml of 10 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA; Sigma), 0.1 mM phenylmethanesulfonyl fluoride (PMSF; Nacalai Tesque), 1 lM pepstatin, and 2 lM leupeptin (Peptide Institute, Osaka, Japan). The homogenates were subsequently centrifuged at 105 000 Â g for 1 h at 4 C. Resulting supernatants were isolated and stored at À80 C. Enzyme-linked immunosorbent assay (ELISA) kits were used to quantify levels of interleukin (IL)-4, and -5 (Thermo Fisher Scientific, Chicago, IL), IL-13 and -33 (R&D Systems, Minneapolis, MN), interferon (IFN) c (e-Bioscience, San Diego, CA) in each supernatant. The total protein level of each cytokine in the lungs was calculated by multiplying the corresponding measured value by the total supernatant volume, as calculated in previous studies (Takano et al. 1997; Yanagisawa et al. 2006) . Kits were also used to assess IL-4, IL-5, IFNc (all e-Bioscience) levels in MLN cell culture supernatants. OVA-specific IgE was measured using a mouse anti-OVA IgE ELISA Kit (Shibayagi Co., Gunma, Japan). Serum OVA-specific IgG 1 was measured as described in Yanagisawa et al. (2006) .
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