pepstatin  (Thermo Fisher)


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    Name:
    Pepstatin A Protease Inhibitor
    Description:
    Thermo Scientific Pepstatin A Protease Inhibitor reversably inhibits aspartic acid proteases Thermo Scientific Protease Inhibitors are small packages of individual protease inhibitor peptides and compounds for customized formulation or modification of protease inhibitor cocktails Features of Thermo Scientific Pepstatin A Protease Inhibitor • High quality reagents in convenient and affordable package sizes • Purchase individually to prepare customized protease inhibitor cocktails • Supplement Halt Protease Inhibitor Cocktails whose formulations are fully disclosed to achieve desired concentrations of specific component reagents The individual reagents include AEBSF aprotinin bestatin E64 leupeptin pepstatin A and PMSF Although Thermo Scientific Halt Protease Inhibitor Cocktails provide convenient and optimized broad spectrum protease inhibition for routine cell lysis and assay needs certain applications require a more customized approach By offering the individual high quality reagents used to make our cocktail products we provide you with the tools needed to apply a protease inhibitor individually supplement a cocktail or combine several components to make customized protease inhibitor cocktails for specialized applications Related Products Pierce Protease and Phosphatase Inhibitor Mini Tablets EDTA Free Pierce Protease Inhibitor Mini Tablets EDTA Free
    Catalog Number:
    78436
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    Cell Lysis & Fractionation|Protease and Phosphatase Inhibition|Protein Biology|Protein Purification & Isolation
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    Structured Review

    Thermo Fisher pepstatin
    Multiplication rates and inhibitor sensitivity. ( A ) WT and FP2KO parasites were cultured in complete medium for three life cycles, and parasitemias were assessed at the end of each cycle. ( B ) Parasites were incubated with various concentrations of chloroquine (Cq), artemisinin (Art), E-64, leupeptin (Leup), and <t>pepstatin</t> (Pep) for one full life cycle, beginning at the ring stage, parasitemias were then determined, and IC 50 values were calculated. Error bars represent the standard deviations of means from three (two for Cq and Art) independent experiments, each performed in duplicate. IC 50 values for WT and FP2KO parasites are shown (μM, except nM for Cq and Art).
    Thermo Scientific Pepstatin A Protease Inhibitor reversably inhibits aspartic acid proteases Thermo Scientific Protease Inhibitors are small packages of individual protease inhibitor peptides and compounds for customized formulation or modification of protease inhibitor cocktails Features of Thermo Scientific Pepstatin A Protease Inhibitor • High quality reagents in convenient and affordable package sizes • Purchase individually to prepare customized protease inhibitor cocktails • Supplement Halt Protease Inhibitor Cocktails whose formulations are fully disclosed to achieve desired concentrations of specific component reagents The individual reagents include AEBSF aprotinin bestatin E64 leupeptin pepstatin A and PMSF Although Thermo Scientific Halt Protease Inhibitor Cocktails provide convenient and optimized broad spectrum protease inhibition for routine cell lysis and assay needs certain applications require a more customized approach By offering the individual high quality reagents used to make our cocktail products we provide you with the tools needed to apply a protease inhibitor individually supplement a cocktail or combine several components to make customized protease inhibitor cocktails for specialized applications Related Products Pierce Protease and Phosphatase Inhibitor Mini Tablets EDTA Free Pierce Protease Inhibitor Mini Tablets EDTA Free
    https://www.bioz.com/result/pepstatin/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pepstatin - by Bioz Stars, 2021-05
    94/100 stars

    Images

    1) Product Images from "Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum"

    Article Title: Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0307720101

    Multiplication rates and inhibitor sensitivity. ( A ) WT and FP2KO parasites were cultured in complete medium for three life cycles, and parasitemias were assessed at the end of each cycle. ( B ) Parasites were incubated with various concentrations of chloroquine (Cq), artemisinin (Art), E-64, leupeptin (Leup), and pepstatin (Pep) for one full life cycle, beginning at the ring stage, parasitemias were then determined, and IC 50 values were calculated. Error bars represent the standard deviations of means from three (two for Cq and Art) independent experiments, each performed in duplicate. IC 50 values for WT and FP2KO parasites are shown (μM, except nM for Cq and Art).
    Figure Legend Snippet: Multiplication rates and inhibitor sensitivity. ( A ) WT and FP2KO parasites were cultured in complete medium for three life cycles, and parasitemias were assessed at the end of each cycle. ( B ) Parasites were incubated with various concentrations of chloroquine (Cq), artemisinin (Art), E-64, leupeptin (Leup), and pepstatin (Pep) for one full life cycle, beginning at the ring stage, parasitemias were then determined, and IC 50 values were calculated. Error bars represent the standard deviations of means from three (two for Cq and Art) independent experiments, each performed in duplicate. IC 50 values for WT and FP2KO parasites are shown (μM, except nM for Cq and Art).

    Techniques Used: Cell Culture, Incubation

    Related Articles

    Activity Assay:

    Article Title: The Inflammatory Response Induced by Aspartic Proteases of Candida albicans Is Independent of Proteolytic Activity ▿
    Article Snippet: Aspartic proteases were purified via anion-exchange chromatography, followed by desalting by passage through a Sephadex G25 column, and lipase was concentrated using Amicon Ultra-15 centrifugal filter units (Millipore). .. The activity of proteases and inhibition by pepstatin A under standard reaction conditions were verified by using a fluorescence-based casein assay (Molecular Probes). ..

    Article Title: Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum
    Article Snippet: .. Parasites were harvested at 6–10% parasitemia as described earlier , and parasite pellets were suspended in 300 μl of PBS [with 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA, 2 mM benzamidine·HCl, 10 μM pepstatin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)] for immunoblot and activity analysis or TRIzol (Invitrogen) for RNA isolation. .. For processing, half of each parasite suspension was solubilized in reducing SDS/PAGE buffer (total lysate) and the other half was subjected to fractionation as described earlier ( ) to prepare total parasite extracts or vacuolar fractions.

    Inhibition:

    Article Title: The Inflammatory Response Induced by Aspartic Proteases of Candida albicans Is Independent of Proteolytic Activity ▿
    Article Snippet: Aspartic proteases were purified via anion-exchange chromatography, followed by desalting by passage through a Sephadex G25 column, and lipase was concentrated using Amicon Ultra-15 centrifugal filter units (Millipore). .. The activity of proteases and inhibition by pepstatin A under standard reaction conditions were verified by using a fluorescence-based casein assay (Molecular Probes). ..

    Fluorescence:

    Article Title: The Inflammatory Response Induced by Aspartic Proteases of Candida albicans Is Independent of Proteolytic Activity ▿
    Article Snippet: Aspartic proteases were purified via anion-exchange chromatography, followed by desalting by passage through a Sephadex G25 column, and lipase was concentrated using Amicon Ultra-15 centrifugal filter units (Millipore). .. The activity of proteases and inhibition by pepstatin A under standard reaction conditions were verified by using a fluorescence-based casein assay (Molecular Probes). ..

    Staining:

    Article Title: Autophagy and ATP-induced anti-apoptosis in antigen presenting cells (APC) follows the cytokine storm in patients after major trauma
    Article Snippet: .. Oceanographic Centre, Southampton, UK) and lysosomes were stained with Pepstatin A or Lysotracker Yellow (Invitrogen.com). .. Confocal scanning microscopy Cell specimens were appropriately labeled with fluorescent dyes and cultured in 15-well-slide™ from Ibidi™ (Ibidi.com).

    Centrifugation:

    Article Title: Cathepsin D Primes Caspase-8 Activation by Multiple Intra-chain Proteolysis *
    Article Snippet: 106 cells were washed with cold PBS supplemented with protease mixture inhibitor and lysed with RIPA buffer (50 m m Tris-HCl, pH 7.4, 150 m m NaCl, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 m m EGTA supplemented with protease inhibitor mixture). .. After centrifugation to remove insoluble particles, equal amounts of cell lysates or 15 μg of cytosolic extracts from freshly isolated human blood neutrophils incubated in the presence or absence of 0.3 units of cathepsin D and PepA (1 μ m ) for 30 min at 37 °C were loaded on gels (NuPAGE, Invitrogen). .. Separated proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore).

    Isolation:

    Article Title: Cathepsin D Primes Caspase-8 Activation by Multiple Intra-chain Proteolysis *
    Article Snippet: 106 cells were washed with cold PBS supplemented with protease mixture inhibitor and lysed with RIPA buffer (50 m m Tris-HCl, pH 7.4, 150 m m NaCl, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 m m EGTA supplemented with protease inhibitor mixture). .. After centrifugation to remove insoluble particles, equal amounts of cell lysates or 15 μg of cytosolic extracts from freshly isolated human blood neutrophils incubated in the presence or absence of 0.3 units of cathepsin D and PepA (1 μ m ) for 30 min at 37 °C were loaded on gels (NuPAGE, Invitrogen). .. Separated proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore).

    Article Title: Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum
    Article Snippet: .. Parasites were harvested at 6–10% parasitemia as described earlier , and parasite pellets were suspended in 300 μl of PBS [with 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA, 2 mM benzamidine·HCl, 10 μM pepstatin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)] for immunoblot and activity analysis or TRIzol (Invitrogen) for RNA isolation. .. For processing, half of each parasite suspension was solubilized in reducing SDS/PAGE buffer (total lysate) and the other half was subjected to fractionation as described earlier ( ) to prepare total parasite extracts or vacuolar fractions.

    Incubation:

    Article Title: Cathepsin D Primes Caspase-8 Activation by Multiple Intra-chain Proteolysis *
    Article Snippet: 106 cells were washed with cold PBS supplemented with protease mixture inhibitor and lysed with RIPA buffer (50 m m Tris-HCl, pH 7.4, 150 m m NaCl, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 m m EGTA supplemented with protease inhibitor mixture). .. After centrifugation to remove insoluble particles, equal amounts of cell lysates or 15 μg of cytosolic extracts from freshly isolated human blood neutrophils incubated in the presence or absence of 0.3 units of cathepsin D and PepA (1 μ m ) for 30 min at 37 °C were loaded on gels (NuPAGE, Invitrogen). .. Separated proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore).

    Western Blot:

    Article Title: Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice
    Article Snippet: For all other data two-tailed unpaired student’s t-tests were used except for the sarkosyl insoluble tau data where we had data from previous studies to inform the predicted direction of change thus allowing the use of a one-tailed test in this case. .. Western blotting Brain tissue was homogenized at 4°C in 9 volumes of buffer (Buffer H) containing (50 mM Tris-HCl (pH 7.4), 20 μM UDP, 0.5 μM PUGNAc, 2 mM sodium othovanadate, 0.1 M NaF, 1.0 mM EGTA, 0.5 mM AEBSF, 2.0 μg/ml aprotinin, 10 μg/ml Leupeptin, 2.0 μg/ml pepstatin A) and the protein concentrations of the homogenates were determined using the BCA method (Pierce). .. The levels of global O -GlcNAcylation (CTD110.6 and RL2) and tau phosphorylation at various sites were determined by Western blot analysis using the antibodies listed in Table .

    BIA-KA:

    Article Title: Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice
    Article Snippet: For all other data two-tailed unpaired student’s t-tests were used except for the sarkosyl insoluble tau data where we had data from previous studies to inform the predicted direction of change thus allowing the use of a one-tailed test in this case. .. Western blotting Brain tissue was homogenized at 4°C in 9 volumes of buffer (Buffer H) containing (50 mM Tris-HCl (pH 7.4), 20 μM UDP, 0.5 μM PUGNAc, 2 mM sodium othovanadate, 0.1 M NaF, 1.0 mM EGTA, 0.5 mM AEBSF, 2.0 μg/ml aprotinin, 10 μg/ml Leupeptin, 2.0 μg/ml pepstatin A) and the protein concentrations of the homogenates were determined using the BCA method (Pierce). .. The levels of global O -GlcNAcylation (CTD110.6 and RL2) and tau phosphorylation at various sites were determined by Western blot analysis using the antibodies listed in Table .

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  • 92
    Thermo Fisher bodipy fl pepstatin a
    P2X7R activation reduced cathepsin D binding and increased lipid oxidation. A ) Stimulation with BzATP (200 μM) inhibited binding of <t>Bodipy</t> FL <t>pepstatin</t> A to active cathepsin D in ARPE-19 cells. * P
    Bodipy Fl Pepstatin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bodipy fl pepstatin a/product/Thermo Fisher
    Average 92 stars, based on 1 article reviews
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    Thermo Fisher pepstatin a
    Effects of heat inactivation of Saps, <t>pepstatin</t> A treatment, and anti-human PAR antibodies on cytokine production induced by Saps. Monocytes were treated with rSap1, rSap2, rSap3, and rSap6 (20 μg/ml) in combination with pepstatin A (15 μM) (A) or with heat-inactivated rSap1, rSap2, rSap3, rSap6, or LPS (1 μg/ml) (B). (C) Effect of pepstatin A (15 μM) or heat inactivation of native or recombinant Sap2 (20 μg/ml) on TNF-α production. After incubation, supernatants were recovered and tested for the presence of TNF-α, IL-1β, or IL-6. Data are expressed as means ± SD for three independent experiments. Statistical analysis was performed with an ANOVA test. *, P
    Pepstatin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    P2X7R activation reduced cathepsin D binding and increased lipid oxidation. A ) Stimulation with BzATP (200 μM) inhibited binding of Bodipy FL pepstatin A to active cathepsin D in ARPE-19 cells. * P

    Journal: The FASEB Journal

    Article Title: Lysosomal alkalinization, lipid oxidation, and reduced phagosome clearance triggered by activation of the P2X7 receptor

    doi: 10.1096/fj.13-236166

    Figure Lengend Snippet: P2X7R activation reduced cathepsin D binding and increased lipid oxidation. A ) Stimulation with BzATP (200 μM) inhibited binding of Bodipy FL pepstatin A to active cathepsin D in ARPE-19 cells. * P

    Article Snippet: After incubation, cells were washed, followed by a 30-min incubation in 10 μM Bodipy FL pepstatin A (Invitrogen) probe at 37°C in the dark.

    Techniques: Activation Assay, Binding Assay

    Pneumolysin progressively translocates from phagolysosomes to the cytosol following challenge with live bacteria. (A) Monocyte-derived macrophages (MDM) were challenged with S. pneumoniae (D39) expressing RFP-tagged pneumolysin (RFP-PLY). At the designated time postchallenge, cells were stained with BODIPY-pepstatin A to visualize lysosomes. Cells were visualized by fluorescence microscopy. Red, PLY; green, lysosomes; yellow, merged. Images are representative of the results of three independent experiments. (B) A Western blot of cytosolic and membrane fractions from D39-exposed MDMs at the designated time points postchallenge probed with anti-pneumolysin (PLY). Actin and lysosome-associated membrane protein 1 (LAMP-1) were used as loading controls. The blots are representative of the results of three independent experiments.

    Journal: mBio

    Article Title: Pneumolysin Activates Macrophage Lysosomal Membrane Permeabilization and Executes Apoptosis by Distinct Mechanisms without Membrane Pore Formation

    doi: 10.1128/mBio.01710-14

    Figure Lengend Snippet: Pneumolysin progressively translocates from phagolysosomes to the cytosol following challenge with live bacteria. (A) Monocyte-derived macrophages (MDM) were challenged with S. pneumoniae (D39) expressing RFP-tagged pneumolysin (RFP-PLY). At the designated time postchallenge, cells were stained with BODIPY-pepstatin A to visualize lysosomes. Cells were visualized by fluorescence microscopy. Red, PLY; green, lysosomes; yellow, merged. Images are representative of the results of three independent experiments. (B) A Western blot of cytosolic and membrane fractions from D39-exposed MDMs at the designated time points postchallenge probed with anti-pneumolysin (PLY). Actin and lysosome-associated membrane protein 1 (LAMP-1) were used as loading controls. The blots are representative of the results of three independent experiments.

    Article Snippet: Macrophages were loaded with 1 µM pepstatin A-BODIPY FL conjugate (Invitrogen) in complete media, for 2 h at 37°C, before being washed in phosphate-buffered saline (PBS) and left overnight.

    Techniques: Derivative Assay, Expressing, Staining, Fluorescence, Microscopy, Western Blot

    Reduced protease density within lysosomes in mutant hAPP Tg neurons. ( A ) Active protease-loaded mature lysosomes are mainly located in the soma of neurons. The mature form of active Cathepsin D (CathD) was labeled by loading Bodipy FL-pepstatin A for 30 min, followed by fixation and immuostaining with antibodies against Cathepsin D or LAMP-1. Bodipy FL-pepstatin A selectively binds to a mature form of active Cathepsin D within mature acidic lysosomes. The co-labeled organelles reflect mature lysosomes (arrows) while puncta labeled by Cathepsin D or LAMP-1 alone are most likely immature Cathepsin D or immature lysosomes (arrowheads). Note that mature lysosomes labeled by both Bodipy FL-pepstatin A and LAMP-1 are barely detected in MAP2-indicated neuronal processes of cortical neurons. ( B–D ) Representative images (B) and quantitative analysis (C and D) showing reduced density of lysosomal Cathepsin D in cortical neurons from mutant hAPP Tg mice. Data were expressed as the mean intensity of LAMP-1 or active Cathepsin D or the co-localized mean intensity of LAMP-1 with active Cathepsin D, respectively. Note that the density of luminal active Cathepsin D within somatic lysosomes positive for both LAMP-1 and Bodipy FL-pepstatin A was significantly reduced in hAPP neurons. ( E and F ) Small hairpin RNA (shRNA)-mediated VPS35 knockdown in WT neurons. Cortical neurons were co-transfected with GFP and VPS35-shRNA or control shRNA at DIV 5, followed by immunostaining with antibodies against VPS35 and MAP2 at DIV10. Arrow points to a neuron with reduced fluorescence intensity of VPS35 in the soma following expressing VPS35-shRNA. The mean intensity of somatic VPS35 was quantified and normalized to that of MAP2 from the same neuron and to those of untransfected neurons from the same imaging fields before comparing to neurons transfected with control shRNA. ( G and H ) Reduced intensity of lysosomal active Cathepsin D labeled by Bodipy FL-pepstatin A in the soma of WT neurons expressing VPS35-shRNA. Quantitative data were expressed as normalized mean intensity of Bodipy FL-pepstatin A fluorescence in the soma of VPS35-shRNA expressed neurons relative to that of controls. Scale bars: 10 μm. Data were quantified from a total number of neurons indicated in parentheses (C) or on the top of bars (D, F and H) from at least three independent experiments. Error bars: SEM. Student's t test: *** P

    Journal: Human Molecular Genetics

    Article Title: Impaired axonal retrograde trafficking of the retromer complex augments lysosomal deficits in Alzheimer’s disease neurons

    doi: 10.1093/hmg/ddx321

    Figure Lengend Snippet: Reduced protease density within lysosomes in mutant hAPP Tg neurons. ( A ) Active protease-loaded mature lysosomes are mainly located in the soma of neurons. The mature form of active Cathepsin D (CathD) was labeled by loading Bodipy FL-pepstatin A for 30 min, followed by fixation and immuostaining with antibodies against Cathepsin D or LAMP-1. Bodipy FL-pepstatin A selectively binds to a mature form of active Cathepsin D within mature acidic lysosomes. The co-labeled organelles reflect mature lysosomes (arrows) while puncta labeled by Cathepsin D or LAMP-1 alone are most likely immature Cathepsin D or immature lysosomes (arrowheads). Note that mature lysosomes labeled by both Bodipy FL-pepstatin A and LAMP-1 are barely detected in MAP2-indicated neuronal processes of cortical neurons. ( B–D ) Representative images (B) and quantitative analysis (C and D) showing reduced density of lysosomal Cathepsin D in cortical neurons from mutant hAPP Tg mice. Data were expressed as the mean intensity of LAMP-1 or active Cathepsin D or the co-localized mean intensity of LAMP-1 with active Cathepsin D, respectively. Note that the density of luminal active Cathepsin D within somatic lysosomes positive for both LAMP-1 and Bodipy FL-pepstatin A was significantly reduced in hAPP neurons. ( E and F ) Small hairpin RNA (shRNA)-mediated VPS35 knockdown in WT neurons. Cortical neurons were co-transfected with GFP and VPS35-shRNA or control shRNA at DIV 5, followed by immunostaining with antibodies against VPS35 and MAP2 at DIV10. Arrow points to a neuron with reduced fluorescence intensity of VPS35 in the soma following expressing VPS35-shRNA. The mean intensity of somatic VPS35 was quantified and normalized to that of MAP2 from the same neuron and to those of untransfected neurons from the same imaging fields before comparing to neurons transfected with control shRNA. ( G and H ) Reduced intensity of lysosomal active Cathepsin D labeled by Bodipy FL-pepstatin A in the soma of WT neurons expressing VPS35-shRNA. Quantitative data were expressed as normalized mean intensity of Bodipy FL-pepstatin A fluorescence in the soma of VPS35-shRNA expressed neurons relative to that of controls. Scale bars: 10 μm. Data were quantified from a total number of neurons indicated in parentheses (C) or on the top of bars (D, F and H) from at least three independent experiments. Error bars: SEM. Student's t test: *** P

    Article Snippet: Sources of antibodies or reagents are as follows: polyclonal antibodies against Cathepsin D (Cat# AF1029, RRID: AB_2087094) (R & D systems), HSP60 (Cat# 4870 S, RRID: AB_2295614) (Cell Signaling Technology), EEA1 (Cat# sc-6416, RRID: AB_640035) and MAP2 (Cat# sc-20172, RRID: AB_2250101) (Santa Cruz), Cathepsin B (Cat# AF965, RRID: AB_2665929), Cathepsin L (Cat# AF1515, RRID: AB_2665930), and VPS35 (Cat# NB100-1397, RRID: AB_527526) (Novus), CI-MPR (Cat# ab124767, RRID: AB_10974087) (Abcam); monoclonal antibodies against LAMP-1 (Cat# 1d4b, RRID: AB_2134500) (Developmental Studies Hybridoma Bank), Neu N (Cat# MAB377, RRID: AB_2298772), DIC (Cat# MAB1618, RRID: AB_2246059), GAPDH (Cat# CB1001, RRID: AB_2107426), and synaptophysin (Cat# MAB5258, RRID: AB_11214133) (Millipore), MAP2 (Cat# 556320, RRID: AB_396359) (BD pharmingen), GM130 (Cat# 610822, RRID: AB_398141) and EGFR (Cat# 610017, RRID: AB_2096701) (BD transduction), synaptophysin (Cat# sc-9116, RRID: AB_2199007) and VPS35 (Cat# 374372, RRID: AB_10988942) (Santa Cruz), VPS26 (Cat# ab181352, RRID: AB_2665924) (Abcam), Rab7 (Cat# R8779, RRID: AB_609910) (Sigma); HA (Cat# 901501, RRID: AB_2565006) (Biolegend); Transferrin receptor (Cat# 13-6800, RRID: AB_2533029), and Alexa fluor 488- (Cat# , RRID: AB_142134) (Cat# , RRID: AB_2534115) (Cat# , RRID: AB_2534102), 546- (Cat# , RRID: AB_2534115) (Cat# , RRID: AB_2534085), and 633- ( , RRID: AB_2535733) (Cat# , RRID: AB_2535720) conjugated secondary antibodies (Invitrogen); Bodipy FL-pepstatin A (Cat# ) (Invitrogen); Magic Red (Cat# 937) (ImmunoChemistry Technologies); control (Cat# sc-108060) and VPS35 shRNA (Cat# sc-63219) (Santa Cruz).

    Techniques: Mutagenesis, Labeling, Mouse Assay, shRNA, Transfection, Immunostaining, Fluorescence, Expressing, Imaging

    Increasing lysosomal delivery of proteases through enhanced retrograde transport of axonal retromer in mutant hAPP Tg neurons. ( A–C ) Representative kymographs (A) and quantitative analysis (B and C) showing enhanced retrograde transport of late endosomal retromer following overexpression of Snapin in mutant hAPP Tg neurons. ( D and E ) Increased density of active Cathepsin D (CathD) labeled by Bodipy FL-pepstatin A in the soma of hAPP neurons expressing Snapin. The mean intensities of Bodipy FL-pepstatin A in the soma of hAPP neurons in the presence and absence of Snapin from the same imaging fields were quantified. Scale bars: 10 μm. Data were quantified from a total number of neurons indicated in parentheses (B and C) or on the top of bars (E) from at least three independent experiments. Error bars represent SEM. Student’s t test: *** p

    Journal: Human Molecular Genetics

    Article Title: Impaired axonal retrograde trafficking of the retromer complex augments lysosomal deficits in Alzheimer’s disease neurons

    doi: 10.1093/hmg/ddx321

    Figure Lengend Snippet: Increasing lysosomal delivery of proteases through enhanced retrograde transport of axonal retromer in mutant hAPP Tg neurons. ( A–C ) Representative kymographs (A) and quantitative analysis (B and C) showing enhanced retrograde transport of late endosomal retromer following overexpression of Snapin in mutant hAPP Tg neurons. ( D and E ) Increased density of active Cathepsin D (CathD) labeled by Bodipy FL-pepstatin A in the soma of hAPP neurons expressing Snapin. The mean intensities of Bodipy FL-pepstatin A in the soma of hAPP neurons in the presence and absence of Snapin from the same imaging fields were quantified. Scale bars: 10 μm. Data were quantified from a total number of neurons indicated in parentheses (B and C) or on the top of bars (E) from at least three independent experiments. Error bars represent SEM. Student’s t test: *** p

    Article Snippet: Sources of antibodies or reagents are as follows: polyclonal antibodies against Cathepsin D (Cat# AF1029, RRID: AB_2087094) (R & D systems), HSP60 (Cat# 4870 S, RRID: AB_2295614) (Cell Signaling Technology), EEA1 (Cat# sc-6416, RRID: AB_640035) and MAP2 (Cat# sc-20172, RRID: AB_2250101) (Santa Cruz), Cathepsin B (Cat# AF965, RRID: AB_2665929), Cathepsin L (Cat# AF1515, RRID: AB_2665930), and VPS35 (Cat# NB100-1397, RRID: AB_527526) (Novus), CI-MPR (Cat# ab124767, RRID: AB_10974087) (Abcam); monoclonal antibodies against LAMP-1 (Cat# 1d4b, RRID: AB_2134500) (Developmental Studies Hybridoma Bank), Neu N (Cat# MAB377, RRID: AB_2298772), DIC (Cat# MAB1618, RRID: AB_2246059), GAPDH (Cat# CB1001, RRID: AB_2107426), and synaptophysin (Cat# MAB5258, RRID: AB_11214133) (Millipore), MAP2 (Cat# 556320, RRID: AB_396359) (BD pharmingen), GM130 (Cat# 610822, RRID: AB_398141) and EGFR (Cat# 610017, RRID: AB_2096701) (BD transduction), synaptophysin (Cat# sc-9116, RRID: AB_2199007) and VPS35 (Cat# 374372, RRID: AB_10988942) (Santa Cruz), VPS26 (Cat# ab181352, RRID: AB_2665924) (Abcam), Rab7 (Cat# R8779, RRID: AB_609910) (Sigma); HA (Cat# 901501, RRID: AB_2565006) (Biolegend); Transferrin receptor (Cat# 13-6800, RRID: AB_2533029), and Alexa fluor 488- (Cat# , RRID: AB_142134) (Cat# , RRID: AB_2534115) (Cat# , RRID: AB_2534102), 546- (Cat# , RRID: AB_2534115) (Cat# , RRID: AB_2534085), and 633- ( , RRID: AB_2535733) (Cat# , RRID: AB_2535720) conjugated secondary antibodies (Invitrogen); Bodipy FL-pepstatin A (Cat# ) (Invitrogen); Magic Red (Cat# 937) (ImmunoChemistry Technologies); control (Cat# sc-108060) and VPS35 shRNA (Cat# sc-63219) (Santa Cruz).

    Techniques: Mutagenesis, Over Expression, Labeling, Expressing, Imaging

    Effects of heat inactivation of Saps, pepstatin A treatment, and anti-human PAR antibodies on cytokine production induced by Saps. Monocytes were treated with rSap1, rSap2, rSap3, and rSap6 (20 μg/ml) in combination with pepstatin A (15 μM) (A) or with heat-inactivated rSap1, rSap2, rSap3, rSap6, or LPS (1 μg/ml) (B). (C) Effect of pepstatin A (15 μM) or heat inactivation of native or recombinant Sap2 (20 μg/ml) on TNF-α production. After incubation, supernatants were recovered and tested for the presence of TNF-α, IL-1β, or IL-6. Data are expressed as means ± SD for three independent experiments. Statistical analysis was performed with an ANOVA test. *, P

    Journal: Infection and Immunity

    Article Title: The Inflammatory Response Induced by Aspartic Proteases of Candida albicans Is Independent of Proteolytic Activity ▿

    doi: 10.1128/IAI.00789-10

    Figure Lengend Snippet: Effects of heat inactivation of Saps, pepstatin A treatment, and anti-human PAR antibodies on cytokine production induced by Saps. Monocytes were treated with rSap1, rSap2, rSap3, and rSap6 (20 μg/ml) in combination with pepstatin A (15 μM) (A) or with heat-inactivated rSap1, rSap2, rSap3, rSap6, or LPS (1 μg/ml) (B). (C) Effect of pepstatin A (15 μM) or heat inactivation of native or recombinant Sap2 (20 μg/ml) on TNF-α production. After incubation, supernatants were recovered and tested for the presence of TNF-α, IL-1β, or IL-6. Data are expressed as means ± SD for three independent experiments. Statistical analysis was performed with an ANOVA test. *, P

    Article Snippet: The activity of proteases and inhibition by pepstatin A under standard reaction conditions were verified by using a fluorescence-based casein assay (Molecular Probes).

    Techniques: Recombinant, Incubation