Cathepsin b

Manufactured by R&D Systems

Sourced in Japan, United States

Cathepsin B is a lysosomal cysteine protease that plays a role in various physiological and pathological processes. It is involved in protein degradation and processing, and has been implicated in conditions such as cancer, inflammation, and neurodegeneration. Cathepsin B is commonly used as a research tool to study these processes.

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Human Pro-Cathepsin B Quantikine ELISA kit Total Human Cathepsin B Quantikine ELISA kit Purified recombinant human cathepsin B Anti-cathepsin B

14 protocols using cathepsin b

Fab Degradation by Cathepsin B and S

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CZP and CZNP were incubated with Cathepsin B (R&D System, Minneapolis, Minnesota) or Cathepsin S (R&D System Minneapolis, Minnesota) with a 10:1 molar ratio (Fab 2µM: Cathepsin 0,2 µM) in the appropriate digestion buffer at varying times at 37° C. Cathepsin B digestion buffer: MES pH5 25mM, 50µM DTT. Cathepsin S digestion buffer: NaOAc 50 mM pH 4.5, 250mM NaCl, 50µM DTT. Cathepsin B and S were activated in the appropriate buffer with 1mM DTT before dilution and use. Reaction was stopped by addition of 5x Laemmli buffer and stored at 4°C before SDS-PAGE analysis. Degradation of Fab was assessed in SDS-PAGE after silver staining of the gels.

de Bourayne M., Meunier S., Bitoun S., Correia E., Mariette X., Nozach H, & Maillère B. (2022). Pegylation Reduces the Uptake of Certolizumab Pegol by Dendritic Cells and Epitope Presentation to T-Cells. Frontiers in Immunology, 13, 808606.

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Targeted Protease Antibody Labeling

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Antibodies toward targeted proteases (cathepsin B, cathepsin L, legumain and human neutrophil elastase) were purchased from R&D Systems. Antibody labeling with stable metal isotopes was performed according to protocol described elsewhere (https://web.stanford.edu/group/nolan/protocols.html). All necessary reagents were purchased from Fluidigm^®.

Poreba M., Groborz K.M., Rut W., Pore M., Snipas S.J., Vizovisek M., Turk B., Kuhn P., Drag M, & Salvesen G.S. (2020). Multiplexed probing of proteolytic enzymes using mass cytometry-compatible activity-based probes.. Journal of the American Chemical Society, 142(39), 16704-16715.

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Pseudovirus Proteolytic Activation Assay

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The proteases used were resuspended according to each of the manufacturer’s protocols: trypsin-TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone) (Sigma), cathepsin L (R&D Systems), cathepsin B (R&D Systems), soluble TMPRSS2 (Abnova), human airway trypsin-like protease (R&D Systems). For each experiment, labeled pseudovirus was diluted 10-fold in HB with the designated protease concentration and incubated at 37°C for 1 h. The mixture was then diluted in 200 μL HB and added to a microfluidic flow cell channel at a rate of 20 μL/min while the video was recorded as described below.

Sengar A., Cervantes M., Bondalapati S.T., Hess T, & Kasson P.M. (2023). Single-Virus Fusion Measurements Reveal Multiple Mechanistically Equivalent Pathways for SARS-CoV-2 Entry. Journal of Virology, 97(5), e01992-22.

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Cathepsin Inhibitors and Viral Infection

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GCDCA and chloroquine were purchased from Sigma-Aldrich (St Louis, MO). Cathepsin L inhibitors, Z-FY-CHO and MDL28170, were purchased from Santa Cruz Biotech (Santa Cruz, CA), and a cathepsin B inhibitor CA074-Me was purchased from Sigma-Aldrich. Recombinant human cathepsin L and cathepsin B were purchased from R & D systems (Minneapolis, MN). The primary antibodies used in this study were anti-PEC/Cowden antibodies raised in swine (Chang et al., 2005 (link)), anti-PEC VP2 antibodies raised in guinea pig (Chang et al., 2005 (link)), anti-FCV antibodies raised in guinea pig (Sosnovtsev and Green, 1995 (link)), anti-MNV-1 antibodies raised in guinea pig (Wobus et al., 2004 (link)) and anti-Rab7 antibodies (Santa Cruz Biotech, CA). The secondary antibodies for Western blot analysis included horseradish peroxidase-conjugates of anti-swine Ig and anti-guinea pig Ig antibody (Thermo Scientific, Pittsburgh, PA). The secondary antibodies for confocal microscopy were FITC-labeled anti-swine IgG for PEC (Kirkegaard & Perry Lab Inc, MD), FITC-labeled anti-guinea pig IgG for MNV-1 (Sigma-Aldrich, MO) and anti-rabbit PerCP-Cy 5.5 (Santa Cruz Biotech, CA). Other basic chemicals for confocal microscopy and other studies were purchased from various sources including Sigma-Aldrich.

Shivanna V., Kim Y, & Chang K.O. (2014). Endosomal acidification and cathepsin L activity is required for calicivirus replication. Virology, 464, 287-295.

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Antibody Sources for Western Blot

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Primary antibodies were obtained from the following sources: Na⁺/K⁺ ATPase‐α, calnexin, calpain 2, CDK2, cyclin B1, cyclin E, ERK2, lamin B, HA tag, p21, p27^Kip1, p57^Kip2, β‐tubulin from Santa Cruz Biotechnology (Santa Cruz, CA), β‐actin from Chemicon International (Billerica, MA), phospho‐AKT (Ser473), AKT, phosphorylated ERK1/2 (T202/Y204), and Lamp1 from Cell Signaling Technology (Danvers, MA) and Cathepsin B from R&D Systems (Mississauga, ON, Canada). Mouse and rabbit horseradish peroxidase antibodies were purchased from Amersham Biosciences (Pittsburg, PA), goat horseradish peroxidase antibody from Santa Cruz, and alkaline phosphatase‐conjugated antibodies were purchased from Promega (Madison, WI).

Bian B., Mongrain S., Cagnol S., Langlois M., Boulanger J., Bernatchez G., Carrier J.C., Boudreau F, & Rivard N. (2015). Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis. Molecular Carcinogenesis, 55(5), 671-687.

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Comprehensive Protein Analysis in Neurobiology

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Antibodies against the following proteins were used in Western blot and immunohistochemical and immunocytochemical analysis: β-Actin (Merck; #A5441); Calbindin (Swant; #300); Cathepsin B (R&D Systems; #AF965); CerS5 (Invitrogen; #PA5-20569); CerS6 (Santa Cruz; #sc100554); EEA1 (R&D Systems; #MAB8047); F4/80 (Abcam; #ab6640); GAPDH (Abcam; #ab8245); GFAP (Merck; # MAB3402); GM130 (BD; #610823); Iba1 (Wako; #019-19741); Lamp1 (DSHB; #1D4B); LSDP5 (Abcam; #ab222811); MAP2 (Biolegend; #822501); PSD95 (Neuromab; #75-028); TOM20 (Santa Cruz; #sc-11415).

Gaudioso Á., Jiang X., Casas J., Schuchman E.H, & Ledesma M.D. (2023). Sphingomyelin 16:0 is a therapeutic target for neuronal death in acid sphingomyelinase deficiency. Cell Death & Disease, 14(4), 248.

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Granzyme B Activation and Inhibition Assay

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Pro-granzyme B (R&D Systems) was activated to granzyme B using cathepsin B (R&D systems) following the manufacturer’s protocol. Enzyme activity was assessed by cleavage of BOC-Ala-Ala-Asp-SBZL (Sigma) and reaction with DTNB (Sigma) with absorbance measurements at 405 nm (NanoDrop 2000 Spectrophotometer, Thermo Scientific). Serial dilutions of non-radioactive gallium-labeled NOTA-GZP were used to inhibit granzyme B by incubation at 37°C for 30 min prior to addition of substrate. Additionally, ⁶⁸Ga-NOTA-GZP was incubated with the activated forms of granzyme B, granzyme A (R&D Systems), granzyme H (R&D Systems), granzyme K (Enzo Life Sciences) and pro-granzyme B to assess the specificity of the peptide. After a 30 min incubation at 37°C, enzymes were purified by size exclusion chromatography and bound radioactivity assessed by gamma counter (Wizard 2480, Perkin Elmer).

Larimer B.M., Wehrenberg-Klee E., Dubois F., Mehta A., Kalomeris T., Flaherty K., Boland G, & Mahmood U. (2017). Granzyme B PET Imaging as a Predictive Biomarker of Immunotherapy Response. Cancer research, 77(9), 2318-2327.

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Protein Analysis in Neurodegenerative Disorders

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Primary antibodies for western blotting were VPS13C (HPA043507, 1:500; Sigma-Aldrich, 28676-1-AP, 1:800; Proteintech), phosphoRab10-T73 (230261, 1:500; Abcam), Rab10 (8127, 1:1,000; Cell Signaling), βIII-Tubulin (4466S, 1:4,000; Biolegend), TH (657012, 1:2,000; Millipore), GAPDH (2,118, 1:2,000; Millipore), α-Tubulin (5168, 1:20,000; Sigma-Aldrich), LAMP2 (H4B4, 1:1,000; DSHB), GCase (G4171, 1:1,000; Sigma-Aldrich), Rab7 (137029, 1:1,000; Abcam), HA (3724, 1:2,000; Cell Signaling), Calnexin (1:1,000; Cell Signaling), TOM20 (612278, 1:1,000; BD Biosciences), PEX5 (83020S, 1:500, Cell Signaling), GFP (1544, 1:2,000; Sigma-Aldrich), Myc (2278, 1:2,000; Cell Signaling), cathepsin B (AF953, 1:2,000; R&D systems), cathepsin D (6487, 1:1,000; Santa Cruz), LRRK2 (ab133474, 1:500; Abcam), LRRK2-S935 (ab133450, 1:500; Abcam), EHBP1 (17637-1-AP, 1:1,000; Proteintech), and PPM1H (PA5-26102, 1:1,000; Invitrogen).
Primary antibodies for immunofluorescence were βIII-Tubulin (4466S, 1:300; Biolegends), TH (657012, 1:300; Millipore), and LAMP1 (sc-20011, 1:300; Santa Cruz).
Primary antibodies for PLA were VPS13C (28676-1-AP, 1:100; Proteintech) and Rab10 (ab104859, 1:100; Abcam).

Schrӧder L.F., Peng W., Gao G., Wong Y.C., Schwake M, & Krainc D. (2024). VPS13C regulates phospho-Rab10-mediated lysosomal function in human dopaminergic neurons. The Journal of Cell Biology, 223(5), e202304042.

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Quantitative Protein Analysis by Western Blot

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Whole cell lysates were prepared as described for quantitative proteome comparison. Cell lysates were centrifuged at 15000 g for 2 min, and protein concentration in the supernatant was determined with a bicinchoninic acid protein assay (BCA, Pierce). Equal amounts of protein extracts (5 to 40 μg depending on the antibody used) were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes using a semi-dry blotting system (Bio-Rad, Hercules, CA, USA). For immune detection of proteins, membranes were probed with the following antibodies: DJ-1 (R&D Systems 3668, Minneapolis, MN, USA), GAP43 (Böhringer 1379011, Ingelheim, Germany), β3-Tubulin (Sigma T8660, Kavasaki, Japan), Cathepsin B (R&D Systems AF953), methylglyoxal (MGO, Cell Biolabs Inc. STA-011, San Diego, CA, USA). β-Tubulin (Sigma Aldrich T6199) or GAPDH (abcam 9484) were used as loading control. methylglyoxal (MGO) immunoblots were stripped by incubating the membrane with pre-heated stripping buffer (2% SDS, 62.5 mM Tris-HCl pH 6.8, 1:125 v/v ß-mercaptoethanol) at 50 °C for 45 min, followed by rinsing for 1–2 h with water and 5 min with TBST before they were blocked and re-probed with GAPDH antibody. Signal intensity was analyzed with ImageJ [38 (link)].

Kern U., Fröhlich K., Bedacht J., Schmidt N., Biniossek M.L., Gensch N., Baerenfaller K, & Schilling O. (2021). Impact of DJ-1 and Helix 8 on the Proteome and Degradome of Neuron-Like Cells. Cells, 10(2), 404.

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Cathepsin B Purification and Characterization

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All reported measurements in this study used purified recombinant human cathepsin B containing proenzyme (∼60%) and mature (∼40%) cathepsin B, purchased from R&D Systems Inc. (Minneapolis, MN, USA). The molecular weights are 37 and 25 kDa, respectively, predicted from the sequence. Thus the average molecular weight of ∼33.8 kDa was used in all concentration calculations. Only a few control experiments in immunoprecipitation study used cathepsin B purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). In normal activity measurements, cathepsin B and the breast cell lysates were activated for 15 min in 5 mM dithiothreitol (DTT) and 25 mM 2-(4-morpholino)ethanesulfonic acid (MES) buffer (pH 5.0) to convert the proenzyme into the active form. The details of other reagents are listed in the Supplementary Information (SI). The synthesis of cathepsin B specific tetrapeptide H₂N-(CH₂)₄-CO-Leu-Arg-Phe-Gly-NH-CH₂-Fc and other peptides including tripeptide Arg-Phe-Gly-NH-CH₂-Fc, dipeptide Phe-Gly-NH-CH₂-Fc and modified single amino acid Gly-NH-CH₂-Fc for studying cleaving sites is also described in the SI (Scheme S1). The cathepsin B inhibitor GC-373 was prepared^{17 (link)} in house as described in the SI.

Swisher L.Z., Prior A.M., Gunaratna M.J., Shishido S., Madiyar F., Nguyen T.A., Hua D.H, & Li J. (2015). Quantitative Electrochemical Detection of Cathepsin B Activity in Breast Cancer Cell Lysates Using Carbon Nanofiber Nanoelectrode Arrays toward Identification of Cancer Formation. Nanomedicine : nanotechnology, biology, and medicine, 11(7), 1695-1704.

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