Cathepsin s

Manufactured by Merck Group

Sourced in United States

Cathepsin S is a proteolytic enzyme that belongs to the papain family of cysteine proteases. It is primarily involved in the degradation of proteins within lysosomes. Cathepsin S plays a role in the processing of antigens for presentation on major histocompatibility complex class II molecules.

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Lab products found in correlation

Cathepsin b, by Merck Group (2 mentions) Cathepsin c, by Merck Group (2 mentions) Cathepsin l, by Merck Group (2 mentions) α actin, by Merck Group (1 mentions) Histone h3, by Abcam (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Ranbp3, by Santa Cruz Biotechnology (1 mentions) α sma, by Cell Signaling Technology (1 mentions) P smad2, by Cell Signaling Technology (1 mentions) P smad3, by Cell Signaling Technology (1 mentions) Z phe arg amc, by Merck Group (1 mentions)

6 protocols using cathepsin s

Immunoblot Analysis of Cathepsin Proteins

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For immunoblot analysis, an equal amount of protein lysate from each cell type preparation or myocardium tissue extract was separated by SDS-PAGE, blotted, and detected with different antibodies, including cathepsin K (CatK) (1:1000, Cat# PB9856, BOSTER, Pleasanton, CA), cathepsin S (CatS) (1:1000) [32 (link)], cathepsin B (CatB) (1:1000, Cat# PC41–100UG, Sigma-Aldrich), cathepsin L (CatL) (1:1000, Cat# 168–10557, RayBiotech, Inc., Norcross, GA), mMCP4 (1:1000), α-SMA (1:1000, Cat# 14968s, Cell Signaling Technology), GAPDH (1:1000, Cat# 2118S, Cell Signaling Technology), p-Smad2 (1:1000, Cat# 3101s, Cell Signaling Technology), p-Smad3 (1:1000, Cat# 9520s, Cell Signaling Technology), importin-β (1:1000, Cat# 8673S, Cell Signaling Technology) and RanBP3 (1:1000, Cat# SC-373678, Santa Cruz Biotechnology, Dallas, TX), α-actin (1:1000, Cat# A5441-.2ML, Sigma-Aldrich), and histone-H3 (1:1000, Cat# ab1791, Abcam).

Wang Y., Liu C.L., Fang W., Zhang X., Yang C., Li J., Liu J., Sukhova G.K., Gurish M.F., Libby P., Shi G.P, & Zhang J. (2019). Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction. Biochimica et biophysica acta. Molecular basis of disease, 1865(6), 1170-1181.

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Inhibitory Activity of Recombinant Protein

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To calculate the inhibitory activity of the recombinant protein, the concentration of rRHcyst-1 at which a 50% inhibition of the proteolytic enzymes’ activities was achieved (IC₅₀) was measured. Recombinant protein was preincubated with each enzyme (0.15 μM) in an assay buffer for 30 min. Then, 0.25 mM of the protease-specific substrates was added to each well and residual enzyme activity monitored [13 (link)]. The GST protein was used as control. Enzymes used were as follows: cathepsin L, C, B, S and H, as well as papain. All of these enzymes were purchased from Sigma Company (St. Louis, USA). The assay buffer used consisted of 100 mM sodium acetate, pH 5.5, 100 mM NaCl, 1 mM EDTA, 1 mg/ml cysteine, and 0.005% TritonX-100. The substrates purchased (Sigma company) were as follows: Z-Phe-Arg-AMC·HCl for papain, cathepsin L and cathepsin B; Pro-Arg-4-methoxy-β-naphthylamide acetate salt for cathepsin C; Arg-NMec·HCl for cathepsin H; and Ac-Lys-Gln-Lys-Leu-Arg-AMC for cathepsin S.

Wang Y., Zhou Y., Gong H., Cao J., Zhang H., Li X, & Zhou J. (2015). Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides. Parasites & Vectors, 8, 140.

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Proteolytic Cleavage of gp120

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Each reaction of 10 μL contained 5 μg of wild-type or variant gp120 and 0, 0.12, 0.18, or 0.24 μg of protease in phosphate-buffered saline (50 mM sodium-phosphate, 125 mM sodium chloride) and 10% glycerol and was incubated as follows. For elastase and trypsin, the reaction mixtures were incubated for 30 min at 37° C and terminated by addition of 1 μL of 100 mM phenylmethylsulfonyl fluoride (PMSF) in ethanol. For cathepsin S (Sigma), reaction mixtures were incubated for 30 min at 25° C and terminated by addition of NuPAGE gel-loading buffer (Invitrogen). For proteinase K, reaction mixtures were incubated for 30 min at 4° C and terminated by addition of 1 μL of 100 mM PMSF as above. Samples were reduced with dithiothreitol, boiled for 2 min, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using a 4 to 12% NuPAGE SDS-morpholineethanesulfonic acid gel (Invitrogen).

Nguyen H.N., Steede N.K., Robinson J.E, & Landry S.J. (2015). Conformational instability governed by disulfide bonds partitions the dominant from subdominant helper T-cell responses specific for HIV-1 envelope glycoprotein gp120. Vaccine, 33(25), 2887-2896.

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Inhibitory Activity of RHcyst-1 Protein

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To calculate the inhibitory activity of RHcyst-1 protein, the IC50 of RHcyst-1 was measured. Purified protein was preincubated with each enzyme (0.15 μM) in a reaction buffer for 30 min. Then, 0.25 mM of the protease-specific substrates was added to each well, and residual enzyme activity was measured [32, 34] . The following enzymes were used: cathepsin B (from bovine spleen), cathepsin C (from bovine spleen), cathepsin L (from human liver) and cathepsin S (from human spleen). These enzymes were all purchased from Sigma Co. (Sigma, USA). The reaction buffer consisted of 100 mM sodium acetate, 100 mM NaCl, 1 mM EDTA, 1 mg/mL cysteine, and 0.005% Triton X-100 [32, 34] . The substrates purchased (Sigma, USA) were as follows: Z-Phe-Arg-AMC•HCl for cathepsin B and cathepsin L; Pro-Arg-4-methoxy-β-naphthylamide acetate salt for cathepsin C and Ac-Lys-Gln-Lys-Leu-Arg-AMC for cathepsin S, and the final concentration of different substrates is 0.25 mM.

Wei N., Lin Z., Xu Z., Cao J., Zhou Y., Zhang H., Gong H., Zhou J, & Li G. (2018). A Tick Cysteine Protease Inhibitor RHcyst-1 Exhibits Antitumor Potential. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 46(6).

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Cathepsin Digestion of Peanut Allergens

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nAra h 6, r/a‐Ara h 6, and rAra h 6 were subjected to cathepsin digestion. Digestion by cathepsin L (Sigma; from human liver, 2.13 U per mg of enzyme) was performed in 400 mM acetate sodium pH 5.5, 4 mM EDTA, and 8 mM DTT. The enzyme:protein ratio was 91 mU:mg and samples were collected at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h (2 μg protein per sampling time). Sample pH was increased to pH 8 by adding sodium bicarbonate and the enzymatic reaction was further stopped using protease inhibitors. Digestion by cathepsin S (Calbiochem‐Merck; human recombinant, 97.55 U per mg of enzyme) was performed in 100 mM acetate sodium pH 5.5, 1 mM EDTA, and 2 mM DTT. The enzyme:protein ratio was 20 mU:mg and samples were collected at 2, 3, 4, 5, 6, 7, and 8 h (2 μg protein per sampling time). Sample pH was increased to pH 8 by adding sodium bicarbonate and the enzymatic reaction was further stopped using protease inhibitors. Samples were analyzed by SDS‐PAGE electrophoresis.
In vitro digestion by trypsin of fractions from roasted peanut obtained by GF was performed as previously described 21. Briefly, digestion was performed with an enzyme:protein ratio of 1:20 (w:w) in 50 mM Tris‐HCl pH 8. Digested fractions were collected at 1, 2, and 4 h. Corresponding samples were analyzed by SDS‐PAGE electrophoresis and anti‐Ara h 6 immunoblot in nonreducing conditions.

Guillon B., Bernard H., Drumare M., Hazebrouck S, & Adel‐Patient K. (2016). Heat processing of peanut seed enhances the sensitization potential of the major peanut allergen Ara h 6. Molecular Nutrition & Food Research, 60(12), 2722-2735.

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Fluorescent Probe Characterization

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For pH dependence experiments, LES12 or LES13 (1 μM) were incubated in buffers of discrete pH made by mixing 0.2 M Na₂HPO₄ and 0.1 M citrate. For activity dependence experiments, 100 nM cathepsin B, cathepsin L (Athens Research & Technology), or 60 nM cathepsin S (EMD Millipore) was preactivated in citrate buffer (50 mM citrate pH 5.5, 0.1% Triton-X-100, 0.5% CHAPS, 5 mM DTT) for 10-30 minutes at 37°C. Activated cathepsin protease was added to pH 5.5 phosphate-citrate buffer containing 1 μM LES12 or LES13. Final probe concentration was 500 nM and final protease concentration was 50 nM. Probe fluorescence was read at 520 nm (490 nm excitation) and 680 nm (650 nm excitation) on a plate reader (BioTek). For quantum yield determination, a range of concentrations of probe or free Cy5 were incubated in citrate buffer. For each concentration of probe, absorbance at 650 nm (measured in transparent 96-well plates in triplicate) and fluorescent emission (680 nm; 650 nm excitation, measured in opaque-walled 96-well plates in triplicate) were measured by plate reader. Fluorescent emission was plotted against absorbance and slopes determined by linear fitting. Quantum yields were calculated using the following equation:
For these experiments, the ‘standard’ is free Cy5 and ‘calc’ is one of the probes.

Sanman L.E., van der Linden W.A., Verdoes M, & Bogyo M. (2016). Bifunctional probes of cathepsin protease activity and pH reveal alterations in endolysosomal pH during bacterial infection. Cell chemical biology, 23(7), 793-804.

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