leupeptin  (Roche)


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    Structured Review

    Roche leupeptin
    Sortilin levels are stabilized by lysosomal inhibition. Serum-starved PC3/GFP-Sortilin cells were incubated with recombinant progranulin (40 nM) with or without <t>leupeptin</t> (100 μM) on ice for 1 h. After washing, cells were shifted to 37 °C and imaged at the indicated time points (seconds). Images were acquired every 12 s for 14 min using live cell confocal microscopy. The background-corrected fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1 and is expressed as a function of time. Error bars represent standard deviation for n = 4 (* P
    Leupeptin, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 419 article reviews
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    92/100 stars

    Images

    1) Product Images from "The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin"

    Article Title: The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2017.04.001

    Sortilin levels are stabilized by lysosomal inhibition. Serum-starved PC3/GFP-Sortilin cells were incubated with recombinant progranulin (40 nM) with or without leupeptin (100 μM) on ice for 1 h. After washing, cells were shifted to 37 °C and imaged at the indicated time points (seconds). Images were acquired every 12 s for 14 min using live cell confocal microscopy. The background-corrected fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1 and is expressed as a function of time. Error bars represent standard deviation for n = 4 (* P
    Figure Legend Snippet: Sortilin levels are stabilized by lysosomal inhibition. Serum-starved PC3/GFP-Sortilin cells were incubated with recombinant progranulin (40 nM) with or without leupeptin (100 μM) on ice for 1 h. After washing, cells were shifted to 37 °C and imaged at the indicated time points (seconds). Images were acquired every 12 s for 14 min using live cell confocal microscopy. The background-corrected fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1 and is expressed as a function of time. Error bars represent standard deviation for n = 4 (* P

    Techniques Used: Inhibition, Incubation, Recombinant, Confocal Microscopy, Fluorescence, Software, Standard Deviation

    2) Product Images from "Role of Proteases in the Release of Porcine Epidemic Diarrhea Virus from Infected Cells ▿"

    Article Title: Role of Proteases in the Release of Porcine Epidemic Diarrhea Virus from Infected Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00464-11

    Inhibition of virus release from PEDV-infected Vero/TMPRSS2 cells after treatment with a serine and cysteine protease inhibitor (leupeptin). Vero and Vero/TMPRSS2 cells were infected with PEDV at an MOI of 0.1. After virus adsorption, cells were washed
    Figure Legend Snippet: Inhibition of virus release from PEDV-infected Vero/TMPRSS2 cells after treatment with a serine and cysteine protease inhibitor (leupeptin). Vero and Vero/TMPRSS2 cells were infected with PEDV at an MOI of 0.1. After virus adsorption, cells were washed

    Techniques Used: Inhibition, Infection, Protease Inhibitor, Adsorption

    Related Articles

    Immunoprecipitation:

    Article Title: The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin
    Article Snippet: .. Lysates from several plates were pooled, and 2 mg of proteins were immunoprecipitated in HNTG buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.2 mM sodium orthovanadate, protease inhibitor mix [Roche]) supplemented with 20 μM of the proteasome inhibitor MG132 (Calbiochem) and 100 μM of leupeptin as lysosomal inhibitors (Roche) to accumulate the ubiquitinated species. .. Filters were immunoblotted with anti-HA monoclonal antibodies (P4D1, Covance) to detect ubiquitinated proteins.

    Protease Inhibitor:

    Article Title: Reduced amyloidogenic processing of the amyloid ?-protein precursor by the small-molecule Differentiation Inducing Factor-1
    Article Snippet: .. Cells were lysed in ice cold RIPA buffer (20 mM Tris–HCl (pH 7.4) containing 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (NP-40), 50 mM NaF, 1 mM Na3 VO4 , 1 mM Na2 MoO4 , 10 mg/mL aprotinin, and 10 mg/mL leupeptin), and supplemented with protease inhibitor tablets (Roche). .. Protein extracts were fractionated by SDS-PAGE and transferred to PVDF membranes.

    Article Title: The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin
    Article Snippet: .. Lysates from several plates were pooled, and 2 mg of proteins were immunoprecipitated in HNTG buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.2 mM sodium orthovanadate, protease inhibitor mix [Roche]) supplemented with 20 μM of the proteasome inhibitor MG132 (Calbiochem) and 100 μM of leupeptin as lysosomal inhibitors (Roche) to accumulate the ubiquitinated species. .. Filters were immunoblotted with anti-HA monoclonal antibodies (P4D1, Covance) to detect ubiquitinated proteins.

    Article Title: Cardiomyocyte S1P1 Receptor–mediated Extracellular Signal–related Kinase Signaling and Desensitization
    Article Snippet: .. Western Blot Analysis For whole cell extraction, cells were lysed in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol-bis(aminoethylether)-tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3 VO4, 1 μg/mL leupeptin, 0.1% protease inhibitor mixture (Roche Applied Science, Indianapolis, IN), and phosphatase inhibitor cocktail (Sigma Chemical, St. Louis, MO). ..

    Concentration Assay:

    Article Title: Role of Proteases in the Release of Porcine Epidemic Diarrhea Virus from Infected Cells ▿
    Article Snippet: .. After another 4 h of incubation at 37°C, DMEM containing 10% TPB and either no leupeptin or the indicated concentration of leupeptin (500, 100, or 20 μM; Roche) were added. .. To determine the cleavability of the S protein by trypsin, Vero cells were infected with PEDV and were incubated without trypsin.

    Incubation:

    Article Title: Role of Proteases in the Release of Porcine Epidemic Diarrhea Virus from Infected Cells ▿
    Article Snippet: .. After another 4 h of incubation at 37°C, DMEM containing 10% TPB and either no leupeptin or the indicated concentration of leupeptin (500, 100, or 20 μM; Roche) were added. .. To determine the cleavability of the S protein by trypsin, Vero cells were infected with PEDV and were incubated without trypsin.

    Inhibition:

    Article Title: Pleiotrophin regulates serine phosphorylation and the cellular distribution of ?-adducin through activation of protein kinase C
    Article Snippet: .. Lysates of cells were prepared in PBS (pH 7.2) with 1% SDS, 1 mM PMSF, 10 mg/ml leupeptin, and 1× Complete Protease Inhibition tablets (Roche Diagnostics), resolved by SDS/PAGE and transferred to a nitrocellulose membrane. ..

    Modification:

    Article Title: The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner
    Article Snippet: .. Treated HL-1 cells were lysed in modified RIPA-buffer (10 mM Na2 HPO4, 150 mM NaCl, 1% Triton-X-100, 0.25% SDS, 1% Na desoxycholate, pH 7.2) supplemented with leupeptin, aprotenin, pepsatin, phenylmethylsulfonyl fluoride, and phosphatase inhibitors (Roche), mixed with Laemmli buffer and loaded on a freshly prepared 6%-polyacrylamide gel containing either 0 mM as control or 30 mM Phos-Tag (ALL-107, Wako Chemicals) to separate phosphorylated proteins. ..

    Western Blot:

    Article Title: STAT3‐dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer
    Article Snippet: .. Western blotting For WB analysis, cells were lysed in RIPA buffer (R0278, Sigma) containing 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 1 μg/ml leupeptin, 10 μg/ml aprotinin, and cOmplete Mini protease inhibitors (11836153001; Roche) or Hunt buffer (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% w/v NP‐40) in the presence of cOmplete protease inhibitors and PhosSTOP™ (Roche). .. 20 μg of each sample was loaded onto a 10% SDS–PAGE minigel (Invitrogen) for electrophoresis.

    Article Title: Cardiomyocyte S1P1 Receptor–mediated Extracellular Signal–related Kinase Signaling and Desensitization
    Article Snippet: .. Western Blot Analysis For whole cell extraction, cells were lysed in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol-bis(aminoethylether)-tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3 VO4, 1 μg/mL leupeptin, 0.1% protease inhibitor mixture (Roche Applied Science, Indianapolis, IN), and phosphatase inhibitor cocktail (Sigma Chemical, St. Louis, MO). ..

    Recombinant:

    Article Title: NADPH oxidase 1 mediates caerulein-induced pancreatic fibrosis in chronic pancreatitis
    Article Snippet: .. Caerulein (#C9026), trypsin inhibitor (SBTI) (#T9003), protease (#P5147) were purchased from Sigma Aldrich (St Louis, MO), collagenase (CLSPA) was purchased from Worthington Biochemical Co. (Lakewood, NJ), collagenase P (11213857001), DNase I recombinant (04716728001), leupeptin and aprotinin were purchased from Roche Diagnostics (Indianapolis, IN), bovine albumin serum fraction V (BSA) from MP Biomedicals (Solon, OH), Phadebas Amylase Assay kit was purchased form Magle Life Sciences (Cambridge, MA), Boc-Gln-Ala-Arg-AMC from BachemAG (Bubendrof, Switzerland), Dulbecco’s minimal essential medium (DMEM)/high glucose from HyClone (Logan, Utah), TRIzol, and 3,3-diaminobenzidine tetrahydrochloride (DAB) from Thermo Scientific (Rockford, IL). .. Protein determination reagent was purchased from Bio-Rad Life Science Research (Hercules, CA).

    SDS Page:

    Article Title: Pleiotrophin regulates serine phosphorylation and the cellular distribution of ?-adducin through activation of protein kinase C
    Article Snippet: .. Lysates of cells were prepared in PBS (pH 7.2) with 1% SDS, 1 mM PMSF, 10 mg/ml leupeptin, and 1× Complete Protease Inhibition tablets (Roche Diagnostics), resolved by SDS/PAGE and transferred to a nitrocellulose membrane. ..

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    Roche leupeptin
    Schematic representation of aSMase processing. A , pro-aSMase (72–75 kDa) arises from prepro-aSMase (75 kDa) within the Golgi and is either targeted to the lysosome via mannose 6-phosphorylation ( M6P ), or is released extracellularly through the default Golgi secretory pathway giving rise to S-SMase (75–80 kDa). Upon entry to the endolysosomal compartment pro-aSMase is cleaved by “protease X” giving rise to the 65-kDa aSMase, which upon encountering Zn 2+ in the lysosome becomes L-SMase. L-SMase (65 kDa) is in turn degraded by “protease Y” to the 52-kDa inactive L-SMase. The action of protease Y is promoted by desipramine, and inhibited by <t>leupeptin.</t> B , both pro-aSMase and S-SMase have an intact C terminus, require exogenous Zn 2+ for activity, and are insensitive to desipramine. Mature L-SMase has lost a portion of its C terminus, which may promote coordination of lysosomal Zn 2+ , thereby rendering the 65-kDa aSMase, Zn 2+ -independent, and is sensitive to desipramine.
    Leupeptin, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/leupeptin/product/Roche
    Average 92 stars, based on 419 article reviews
    Price from $9.99 to $1999.99
    leupeptin - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    Image Search Results


    Schematic representation of aSMase processing. A , pro-aSMase (72–75 kDa) arises from prepro-aSMase (75 kDa) within the Golgi and is either targeted to the lysosome via mannose 6-phosphorylation ( M6P ), or is released extracellularly through the default Golgi secretory pathway giving rise to S-SMase (75–80 kDa). Upon entry to the endolysosomal compartment pro-aSMase is cleaved by “protease X” giving rise to the 65-kDa aSMase, which upon encountering Zn 2+ in the lysosome becomes L-SMase. L-SMase (65 kDa) is in turn degraded by “protease Y” to the 52-kDa inactive L-SMase. The action of protease Y is promoted by desipramine, and inhibited by leupeptin. B , both pro-aSMase and S-SMase have an intact C terminus, require exogenous Zn 2+ for activity, and are insensitive to desipramine. Mature L-SMase has lost a portion of its C terminus, which may promote coordination of lysosomal Zn 2+ , thereby rendering the 65-kDa aSMase, Zn 2+ -independent, and is sensitive to desipramine.

    Journal: The Journal of Biological Chemistry

    Article Title: A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

    doi: 10.1074/jbc.M110.155234

    Figure Lengend Snippet: Schematic representation of aSMase processing. A , pro-aSMase (72–75 kDa) arises from prepro-aSMase (75 kDa) within the Golgi and is either targeted to the lysosome via mannose 6-phosphorylation ( M6P ), or is released extracellularly through the default Golgi secretory pathway giving rise to S-SMase (75–80 kDa). Upon entry to the endolysosomal compartment pro-aSMase is cleaved by “protease X” giving rise to the 65-kDa aSMase, which upon encountering Zn 2+ in the lysosome becomes L-SMase. L-SMase (65 kDa) is in turn degraded by “protease Y” to the 52-kDa inactive L-SMase. The action of protease Y is promoted by desipramine, and inhibited by leupeptin. B , both pro-aSMase and S-SMase have an intact C terminus, require exogenous Zn 2+ for activity, and are insensitive to desipramine. Mature L-SMase has lost a portion of its C terminus, which may promote coordination of lysosomal Zn 2+ , thereby rendering the 65-kDa aSMase, Zn 2+ -independent, and is sensitive to desipramine.

    Article Snippet: Leupeptin, pepstatin, and aprotinin were obtained from Roche Applied Science.

    Techniques: Activity Assay

    Complex formation of PLD1 with caveolin-1 and with the EGFR in the CEM. COS-7 cells transfected with WT or C240S/C241S mutant of PLD1 were incubated in the presence or absence of 100 nM of EGF for 0.5 min. The CEM fraction was prepared, and the proteins were solubilized in an extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Nonidet P-40, 0.4% deoxycholate, 60 mM β-octylglucopyranoside, 0.5 mM phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, and 5 μg/ml aprotinin). The sample was centrifuged at 150,000 × g for 1 h, and the solubilized supernatant was incubated with anti-PLD antibody precoupled to protein A agarose resin. The immune complexes were subjected to 6–16% gradient SDS-PAGE followed by immunoblot analysis with anti-PLD, anti-EGFR, or anti-caveolin-1 antibody. The blots shown are representative of three separate results. I.P., immunoprecipitate.

    Journal: Molecular Biology of the Cell

    Article Title: Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

    doi: 10.1091/mbc.E02-02-0100

    Figure Lengend Snippet: Complex formation of PLD1 with caveolin-1 and with the EGFR in the CEM. COS-7 cells transfected with WT or C240S/C241S mutant of PLD1 were incubated in the presence or absence of 100 nM of EGF for 0.5 min. The CEM fraction was prepared, and the proteins were solubilized in an extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Nonidet P-40, 0.4% deoxycholate, 60 mM β-octylglucopyranoside, 0.5 mM phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, and 5 μg/ml aprotinin). The sample was centrifuged at 150,000 × g for 1 h, and the solubilized supernatant was incubated with anti-PLD antibody precoupled to protein A agarose resin. The immune complexes were subjected to 6–16% gradient SDS-PAGE followed by immunoblot analysis with anti-PLD, anti-EGFR, or anti-caveolin-1 antibody. The blots shown are representative of three separate results. I.P., immunoprecipitate.

    Article Snippet: Phenylmethylsulfonylfluoride, leupeptin, and aprotinin were obtained from Roche Molecular Biochemicals; paraformaldehyde and anti-actin antibody from Sigma (St. Louis, MO); [3 H]palmitic acid and [32 P]orthophosphate from Dupont NEN (Boston, MA); [3 H]myristic acid and the chemiluminescence kit (ECL system) from Amersham International (Buckinghamshire, U.K.); Silica Gel 60 TLC plates from Merck (Darmstadt, Germany); immobilized protein A and rhodamine-conjugated anti-mouse antibody from Pierce (Rockford, IL); DMEM and LipofectAmine from Gibco-BRL (Grand Island, NY); fetal bovine serum from PAA Laboratories, Inc. (Parker Ford, PA); and horseradish peroxidase–conjugated goat anti-rabbit IgG or anti-mouse IgG, IgM, and IgA from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD).

    Techniques: Transfection, Mutagenesis, Incubation, SDS Page

    Tetraspanin effects on MT1-MMP-dependent fibronectin degradation. MCF-7-VC and MCF-7-MT1 cells were treated with siRNAs, plated 4 d later on fibronectin (FN)-coated slides, and then incubated overnight in serum-free media with protease inhibitors (50 μg/ml aprotinin, 2 μM leupeptin, 20 μM E64, and 20 μM pepstatin A) to prevent non-MMP proteolysis of the FN. The following day, cells were washed, fixed, and FN was detected using an anti-FN antibody followed by Alexa 488-conjugated anti-mouse secondary antibody (green). The position of the cells was determined by staining the cytoskeleton with Alexa Fluor 546-phalloidin (red) and the nuclei with DAPI (blue). Representative fluorescent images (merge green/red/blue or green only) are shown. Bar, 20 μm. For the bar graph, 100% fibronectin degradation is defined as 0 pixel density within defined proteolyzed areas, whereas 0% degradation is pixel density in the unperturbed fibronectin layer. Results are mean of percentage of fibronectin degradation ± SD (n = 3; *p

    Journal: Molecular Biology of the Cell

    Article Title: Tetraspanin Proteins Regulate Membrane Type-1 Matrix Metalloproteinase-dependent Pericellular Proteolysis

    doi: 10.1091/mbc.E08-11-1149

    Figure Lengend Snippet: Tetraspanin effects on MT1-MMP-dependent fibronectin degradation. MCF-7-VC and MCF-7-MT1 cells were treated with siRNAs, plated 4 d later on fibronectin (FN)-coated slides, and then incubated overnight in serum-free media with protease inhibitors (50 μg/ml aprotinin, 2 μM leupeptin, 20 μM E64, and 20 μM pepstatin A) to prevent non-MMP proteolysis of the FN. The following day, cells were washed, fixed, and FN was detected using an anti-FN antibody followed by Alexa 488-conjugated anti-mouse secondary antibody (green). The position of the cells was determined by staining the cytoskeleton with Alexa Fluor 546-phalloidin (red) and the nuclei with DAPI (blue). Representative fluorescent images (merge green/red/blue or green only) are shown. Bar, 20 μm. For the bar graph, 100% fibronectin degradation is defined as 0 pixel density within defined proteolyzed areas, whereas 0% degradation is pixel density in the unperturbed fibronectin layer. Results are mean of percentage of fibronectin degradation ± SD (n = 3; *p

    Article Snippet: Protein G-agarose beads, aprotinin, and leupeptin (serine and thiol protease inhibitor) were from Roche Diagnostics.

    Techniques: Incubation, Staining

    Sortilin levels are stabilized by lysosomal inhibition. Serum-starved PC3/GFP-Sortilin cells were incubated with recombinant progranulin (40 nM) with or without leupeptin (100 μM) on ice for 1 h. After washing, cells were shifted to 37 °C and imaged at the indicated time points (seconds). Images were acquired every 12 s for 14 min using live cell confocal microscopy. The background-corrected fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1 and is expressed as a function of time. Error bars represent standard deviation for n = 4 (* P

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin

    doi: 10.1016/j.matbio.2017.04.001

    Figure Lengend Snippet: Sortilin levels are stabilized by lysosomal inhibition. Serum-starved PC3/GFP-Sortilin cells were incubated with recombinant progranulin (40 nM) with or without leupeptin (100 μM) on ice for 1 h. After washing, cells were shifted to 37 °C and imaged at the indicated time points (seconds). Images were acquired every 12 s for 14 min using live cell confocal microscopy. The background-corrected fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1 and is expressed as a function of time. Error bars represent standard deviation for n = 4 (* P

    Article Snippet: Lysates from several plates were pooled, and 2 mg of proteins were immunoprecipitated in HNTG buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.2 mM sodium orthovanadate, protease inhibitor mix [Roche]) supplemented with 20 μM of the proteasome inhibitor MG132 (Calbiochem) and 100 μM of leupeptin as lysosomal inhibitors (Roche) to accumulate the ubiquitinated species.

    Techniques: Inhibition, Incubation, Recombinant, Confocal Microscopy, Fluorescence, Software, Standard Deviation