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  • 97
    Millipore pepstatin
    Aspartic protease purification and activity. Fig 1A. Coomassie stained PAGE gel showing fractions eluted from <t>pepstatin</t> affinity purification from the M44 strain. The purified aspartic proteases run around 42 kDa. Fraction numbers are listed above the lanes. Fig 1B. Commassie stained PAGE gel showing aspartic protease degradation of human IgG with and without pepstatin A inhibitor (100 μM). Purified fraction #3 (F3) from the M44 strain was incubated with IgG in sodium citrate buffer at pH 5.5 for 20 hours at 37°C. Nonreduced gel showing the whole assembled antibody and degradation products. The fully assembled antibody runs above 150 kDa. Fig 1C. Commassie stained PAGE gel visualizing the peak fractions from aspartic protease purifications done on the M169 parental strain and M182 Δ pep1 strain. The control aspartic protease sample was from a previous purification. The predominant band at 42 kDa corresponds to pep1 .
    Pepstatin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 4764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam pepstatin
    Aspartic protease purification and activity. Fig 1A. Coomassie stained PAGE gel showing fractions eluted from <t>pepstatin</t> affinity purification from the M44 strain. The purified aspartic proteases run around 42 kDa. Fraction numbers are listed above the lanes. Fig 1B. Commassie stained PAGE gel showing aspartic protease degradation of human IgG with and without pepstatin A inhibitor (100 μM). Purified fraction #3 (F3) from the M44 strain was incubated with IgG in sodium citrate buffer at pH 5.5 for 20 hours at 37°C. Nonreduced gel showing the whole assembled antibody and degradation products. The fully assembled antibody runs above 150 kDa. Fig 1C. Commassie stained PAGE gel visualizing the peak fractions from aspartic protease purifications done on the M169 parental strain and M182 Δ pep1 strain. The control aspartic protease sample was from a previous purification. The predominant band at 42 kDa corresponds to pep1 .
    Pepstatin, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LKT Laboratories pepstatin a
    Enhanced growth inhibition effect of the vacuolar (H + )‐ ATP ase inhibitor by pretreatment with a cathepsin D inhibitor or  CTSD  knockdown. (a,b )  RCC 4 renal cell carcinoma cells plus vector alone ( RCC 4‐vec) and  RKO  colon cancer cells were treated with the indicated concentrations of pepstatin A. After 1 day, these cells were treated with the indicated concentrations of bafilomycin A1. After 3 days, cell viability was assessed. Ordinate values were obtained by setting the control group value as 100%. Data are presented as mean ±  SD  ( n  = 3). * P
    Pepstatin A, supplied by LKT Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris pepstatin a
    Amino acid sequence encoded by exon 4 determines APOL1 cytotoxicity. A : fluorescence microscopy images of HEK293T cells transfected with a control vector (pcDNA3.1) or vectors expressing APOL1 splice variants or deletion mutants and stained 24 h later with the DNA dyes Hoechst 33342 (blue) and propidium iodide (PI; red). Scale bars = 100 μm. B : HEK293T cells were transfected in triplicates with APOL1 expression vectors or control pcDNA3.1 (solid bars) and cultured in medium supplemented with 10% FCS. To analyze whether lysosomal damage contributes to cytotoxicity, at 5 h after transfection the culture medium was replaced with medium supplemented with 2% FCS, and cells were treated with the lysosomal protease inhibitors E64d and <t>pepstatin</t> A (denoted E64d) (hatched bars). At 24 h after transfection, cell lysates and culture supernatants were collected. Culture supernatants were subjected to lactate dehydrogenase (LDH) assay. Cytotoxicity of APOL1 variants is expressed relative to completely lysed control cells, which is set as 100%. Values are means ± SD of 3 independent samples. * P
    Pepstatin A, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Selleck Chemicals pepstatin a
    Expression of IL-8 after pepsin exposure. a Change in inflammatory cytokine expression of Hep-2 and Tu212 cells exposed to different concentrations of pepsin measured using the CBA assay. Production of IL-8 and IL-6 by Hep-2 and Tu212 cells at different pepsin concentrations. b Change in inflammatory cytokine expression of Hep-2 and Tu212 cells exposed to pepsin with/without <t>pepstatin</t> measured using CBA assays. Production of IL-8 and IL-6 by Hep-2 and Tu212 cells at different concentrations of pepsin. c Photomicrographs representative of the immunohistochemical analyses of pepsin and IL-8 in tissue specimens from three patients with laryngeal carcinoma (magnification, ×400). * P
    Pepstatin A, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pepstatin a pepstatin a
    Impact of <t>Pepstatin</t> A on neutrophil migration induced by patient gastric fluid samples set at pH 3 and pH 7.4. Pepstatin A (20 µg/ml) was added to gastric samples set at pH 3 and pH 7.4. Pepstatin A failed to significantly reduce the magnitude of neutrophil (PMN) migration across H292 lung epithelium induced by gastric samples set at pH 3 from patients off PPI therapy ( A ), but did significantly block neutrophil migration induced by gastric samples set at pH 3 from patients on PPI therapy ( B ). For gastric fluid samples set at pH 7.4, Pepstatin A failed to impact the magnitude of neutrophil migration induced by samples collected from patients off ( C ) and on ( D ) PPI therapy. A dilution of 1:4 in pH matched HBSS was used for all samples. Both on and off PPI therapy groups set at either pH 3 or 7.4 represent the mean +/− SEM of the average magnitude values (n = 3) of PMN migration by gastric fluid derived from 12 patients on PPI therapy and 12 patients off PPI therapy, diluted 1:4 in HBSS and treated with or without Pepstatin A (n = 12). Differences were considered significant between groups with and without Pepstatin A at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. Panels A–D are representative internally controlled experiments conducted on at least two separate occasions yielding similar results.
    Pepstatin A Pepstatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Adipogen pepstatin
    Impact of <t>Pepstatin</t> A on neutrophil migration induced by patient gastric fluid samples set at pH 3 and pH 7.4. Pepstatin A (20 µg/ml) was added to gastric samples set at pH 3 and pH 7.4. Pepstatin A failed to significantly reduce the magnitude of neutrophil (PMN) migration across H292 lung epithelium induced by gastric samples set at pH 3 from patients off PPI therapy ( A ), but did significantly block neutrophil migration induced by gastric samples set at pH 3 from patients on PPI therapy ( B ). For gastric fluid samples set at pH 7.4, Pepstatin A failed to impact the magnitude of neutrophil migration induced by samples collected from patients off ( C ) and on ( D ) PPI therapy. A dilution of 1:4 in pH matched HBSS was used for all samples. Both on and off PPI therapy groups set at either pH 3 or 7.4 represent the mean +/− SEM of the average magnitude values (n = 3) of PMN migration by gastric fluid derived from 12 patients on PPI therapy and 12 patients off PPI therapy, diluted 1:4 in HBSS and treated with or without Pepstatin A (n = 12). Differences were considered significant between groups with and without Pepstatin A at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. Panels A–D are representative internally controlled experiments conducted on at least two separate occasions yielding similar results.
    Pepstatin, supplied by Adipogen, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Applichem pepstatin a
    Impact of <t>Pepstatin</t> A on neutrophil migration induced by patient gastric fluid samples set at pH 3 and pH 7.4. Pepstatin A (20 µg/ml) was added to gastric samples set at pH 3 and pH 7.4. Pepstatin A failed to significantly reduce the magnitude of neutrophil (PMN) migration across H292 lung epithelium induced by gastric samples set at pH 3 from patients off PPI therapy ( A ), but did significantly block neutrophil migration induced by gastric samples set at pH 3 from patients on PPI therapy ( B ). For gastric fluid samples set at pH 7.4, Pepstatin A failed to impact the magnitude of neutrophil migration induced by samples collected from patients off ( C ) and on ( D ) PPI therapy. A dilution of 1:4 in pH matched HBSS was used for all samples. Both on and off PPI therapy groups set at either pH 3 or 7.4 represent the mean +/− SEM of the average magnitude values (n = 3) of PMN migration by gastric fluid derived from 12 patients on PPI therapy and 12 patients off PPI therapy, diluted 1:4 in HBSS and treated with or without Pepstatin A (n = 12). Differences were considered significant between groups with and without Pepstatin A at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. Panels A–D are representative internally controlled experiments conducted on at least two separate occasions yielding similar results.
    Pepstatin A, supplied by Applichem, used in various techniques. Bioz Stars score: 92/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioShop pepstatin a
    Impact of <t>Pepstatin</t> A on neutrophil migration induced by patient gastric fluid samples set at pH 3 and pH 7.4. Pepstatin A (20 µg/ml) was added to gastric samples set at pH 3 and pH 7.4. Pepstatin A failed to significantly reduce the magnitude of neutrophil (PMN) migration across H292 lung epithelium induced by gastric samples set at pH 3 from patients off PPI therapy ( A ), but did significantly block neutrophil migration induced by gastric samples set at pH 3 from patients on PPI therapy ( B ). For gastric fluid samples set at pH 7.4, Pepstatin A failed to impact the magnitude of neutrophil migration induced by samples collected from patients off ( C ) and on ( D ) PPI therapy. A dilution of 1:4 in pH matched HBSS was used for all samples. Both on and off PPI therapy groups set at either pH 3 or 7.4 represent the mean +/− SEM of the average magnitude values (n = 3) of PMN migration by gastric fluid derived from 12 patients on PPI therapy and 12 patients off PPI therapy, diluted 1:4 in HBSS and treated with or without Pepstatin A (n = 12). Differences were considered significant between groups with and without Pepstatin A at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. Panels A–D are representative internally controlled experiments conducted on at least two separate occasions yielding similar results.
    Pepstatin A, supplied by BioShop, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim pepstatin a
    Western blot of sLOX-1 released from cells treated with MβCD and different protease inhibitors. LOX-1-V5-COS transfected cells were incubated with MβCD, GM6001, PMSF, <t>Pepstatin</t> A and the protease inhibitor cocktail (PI mix) (Calbiochem), as indicated (upper panel). Histogram shows the densitometric analysis expressed as percentage of reduction of sLOX-1 released in the presence of different protease inhibitors. 100% refers as sLOX-1 released in the presence of 5mM MβCD for 30 min (lower panel). Data in histograms represent the average ± SEM of three experiments, P
    Pepstatin A, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific pepstatin a
    WNV-NY replication does not induce autophagy in 293T cells. (A) Effect of protease inhibitors on WNV replication. 293T cells infected with WNV-NY (MOI = 3) were incubated in the presence (Prot. Inhibit.) or absence (None) of 10 µg/mL <t>Pepstatin</t> A and E64d. Media was removed from cultures at the indicated times, cleared of cell debris, and titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from two independent experiments. (B) Effect of WNV-NY infection on steady-state levels of LC3B-II. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3). After infection, the inoculum was replaced with medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV-NY infection on steady-state levels of LC3B-II and p62. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), GAPDH (third panel), and WNV (bottom panel). (D) Effect of WNV infection of levels of p62. 293T cells were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of p62 (top), GAPDH (middle) or WNV (bottom).
    Pepstatin A, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare pepstatin a
    WNV-NY replication does not induce autophagy in 293T cells. (A) Effect of protease inhibitors on WNV replication. 293T cells infected with WNV-NY (MOI = 3) were incubated in the presence (Prot. Inhibit.) or absence (None) of 10 µg/mL <t>Pepstatin</t> A and E64d. Media was removed from cultures at the indicated times, cleared of cell debris, and titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from two independent experiments. (B) Effect of WNV-NY infection on steady-state levels of LC3B-II. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3). After infection, the inoculum was replaced with medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV-NY infection on steady-state levels of LC3B-II and p62. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), GAPDH (third panel), and WNV (bottom panel). (D) Effect of WNV infection of levels of p62. 293T cells were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of p62 (top), GAPDH (middle) or WNV (bottom).
    Pepstatin A, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 242 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Peninsula Laboratories pepstatin a
    WNV-NY replication does not induce autophagy in 293T cells. (A) Effect of protease inhibitors on WNV replication. 293T cells infected with WNV-NY (MOI = 3) were incubated in the presence (Prot. Inhibit.) or absence (None) of 10 µg/mL <t>Pepstatin</t> A and E64d. Media was removed from cultures at the indicated times, cleared of cell debris, and titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from two independent experiments. (B) Effect of WNV-NY infection on steady-state levels of LC3B-II. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3). After infection, the inoculum was replaced with medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV-NY infection on steady-state levels of LC3B-II and p62. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), GAPDH (third panel), and WNV (bottom panel). (D) Effect of WNV infection of levels of p62. 293T cells were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of p62 (top), GAPDH (middle) or WNV (bottom).
    Pepstatin A, supplied by Peninsula Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Peptide Institute pepstatin a
    Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), <t>pepstatin</t> A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
    Pepstatin A, supplied by Peptide Institute, used in various techniques. Bioz Stars score: 92/100, based on 409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Peptides International pepstatin
    Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), <t>pepstatin</t> A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.
    Pepstatin, supplied by Peptides International, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche pepstatin
    Ex vivo invasive activity of VSMCs in 3-D collagen. (a–c) Media explants of mouse aorta were embedded within a 3-D gel of type I collagen, VSMC egress was initiated by exogenous PDGF/FGF-2, and outgrowth into the translucent collagen matrix was visualized by darkfield microscopy at 0, 3, and 8 d. Asterisk indicates explant , and arrowheads point at the leading edge of the invading SMC. Bar, 1 mm. Inset in (a) shows anti-SM α-actin staining (brown) of the media explant. (d and e) Scanning electron micrograph of freeze-fractured explant cultures. Asterisks mark invading VSMCs that have infiltrated into the field of densely packed type I collagen fibrils. Arrows highlight VSMC cytoplasmic extensions. Tunnels (star) in the collagen matrix are seen in areas surrounding the embedded explant. Bar, 20 μm. (f) Degraded collagen (detected by mAb HUI77) appears as punctate green staining (arrows) surrounding propidium iodide–labeled cells (red, asterisks). Bar, 20 μm. (g–o) Media explants from WT, plasminogen (plg)-, MMP-9–, or MMP-2–null mice were suspended in collagen in autologous sera and cultured for 8 d in the absence or presence of E-64, <t>pepstatin,</t> aprotinin, BB-94, TIMP-1, or TIMP-2 as described in Materials and methods. Insets in j, k, n, and o show immunostains for degraded collagen (mAb HUI77) surrounding propidium iodide–labeled cells. Bar, 1 mm. (p) Quantitative analysis of VSMC invasive activity in aortic explant cultures after an 8-d culture period. Results are expressed as the mean ± SEM ( n = 5).
    Pepstatin, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 2025 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical pepstatin a
    Effect of TXA1 on the expression levels of LC3-II in A375-C5 cells. Cells were treated for 48 h with medium (blank), TXA1 (3.6 μM or 7.2 μM), or with the corresponding DMSO concentrations (DMSO or DMSO2, respectively) LC3-II protein levels were analyzed by Western blot. ( A ) Following treatment with TXA1 alone; ( B ) following co-treatment with 3-MA; and ( C ) following co-treatment with <t>E-64d/pepstatin.</t> Images are representative of, at least, three independent experiments (except for the case of blank and DMSO treatments in the presence of E-64d/pepstatin, which result from two experiments only). Results of the densitometry analysis are expressed after normalization of the values obtained for each protein with the values obtained for tubulin or actin (and further expressed in relation to blank cells) and represent the mean ± SEM from, at least, three independent experiments (except for the case of blank and DMSO treatments in the presence of E-64d/pepstatin, which result from two experiments only). * p ≤ 0.05 Blank vs. treatment.
    Pepstatin A, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Aspartic protease purification and activity. Fig 1A. Coomassie stained PAGE gel showing fractions eluted from pepstatin affinity purification from the M44 strain. The purified aspartic proteases run around 42 kDa. Fraction numbers are listed above the lanes. Fig 1B. Commassie stained PAGE gel showing aspartic protease degradation of human IgG with and without pepstatin A inhibitor (100 μM). Purified fraction #3 (F3) from the M44 strain was incubated with IgG in sodium citrate buffer at pH 5.5 for 20 hours at 37°C. Nonreduced gel showing the whole assembled antibody and degradation products. The fully assembled antibody runs above 150 kDa. Fig 1C. Commassie stained PAGE gel visualizing the peak fractions from aspartic protease purifications done on the M169 parental strain and M182 Δ pep1 strain. The control aspartic protease sample was from a previous purification. The predominant band at 42 kDa corresponds to pep1 .

    Journal: PLoS ONE

    Article Title: Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

    doi: 10.1371/journal.pone.0134723

    Figure Lengend Snippet: Aspartic protease purification and activity. Fig 1A. Coomassie stained PAGE gel showing fractions eluted from pepstatin affinity purification from the M44 strain. The purified aspartic proteases run around 42 kDa. Fraction numbers are listed above the lanes. Fig 1B. Commassie stained PAGE gel showing aspartic protease degradation of human IgG with and without pepstatin A inhibitor (100 μM). Purified fraction #3 (F3) from the M44 strain was incubated with IgG in sodium citrate buffer at pH 5.5 for 20 hours at 37°C. Nonreduced gel showing the whole assembled antibody and degradation products. The fully assembled antibody runs above 150 kDa. Fig 1C. Commassie stained PAGE gel visualizing the peak fractions from aspartic protease purifications done on the M169 parental strain and M182 Δ pep1 strain. The control aspartic protease sample was from a previous purification. The predominant band at 42 kDa corresponds to pep1 .

    Article Snippet: Chemical list PMSF (Sigma #P7626), chymostatin (Sigma #C7268), pepstatin (Sigma #P5318), E-64 (Sigma #E3121), EDTA, human IgG (Sigma #I2511).

    Techniques: Purification, Activity Assay, Staining, Polyacrylamide Gel Electrophoresis, Affinity Purification, Incubation

    In vitro studies to delineate the role of cathepsin G in the proteolysis of band 3. Heat-stressed normal (N) erythrocytes were incubated with IgG-depleted/complement-inactivated sera from the same individual and also with sera from CML patients designated C (N2 with C6, N3 with C10 and N5 with C13) at 37°C for 20 min. Western blots prepared from the lysates of these erythrocytes were stained with cathepsin G and band 3 antibodies. (a) Cathepsin G levels are higher in the membrane of erythrocytes incubated with CML sera as compared to normal sera. (b) Staining of the same blot with anti-band 3 antibody shows that a 20 kDa fragment of band 3 is seen in cells incubated with CML sera. (c) The generation of 20 kDa fragment is diminished upon incubation with pepstatin A (pep) and is nearly inhibited by cathepsin G inhibitor 1 (CI). Densitometric readings of each lane of the band 3 autograph corresponding to the lanes in (c) demonstrate equal loading.

    Journal: Anemia

    Article Title: Eryptotic Phenotype in Chronic Myeloid Leukemia: Contribution of Neutrophilic Cathepsin G

    doi: 10.1155/2012/659303

    Figure Lengend Snippet: In vitro studies to delineate the role of cathepsin G in the proteolysis of band 3. Heat-stressed normal (N) erythrocytes were incubated with IgG-depleted/complement-inactivated sera from the same individual and also with sera from CML patients designated C (N2 with C6, N3 with C10 and N5 with C13) at 37°C for 20 min. Western blots prepared from the lysates of these erythrocytes were stained with cathepsin G and band 3 antibodies. (a) Cathepsin G levels are higher in the membrane of erythrocytes incubated with CML sera as compared to normal sera. (b) Staining of the same blot with anti-band 3 antibody shows that a 20 kDa fragment of band 3 is seen in cells incubated with CML sera. (c) The generation of 20 kDa fragment is diminished upon incubation with pepstatin A (pep) and is nearly inhibited by cathepsin G inhibitor 1 (CI). Densitometric readings of each lane of the band 3 autograph corresponding to the lanes in (c) demonstrate equal loading.

    Article Snippet: Chemicals Pepstatin A (P 4265), phenyl methyl sulfonyl fluoride (PMSF) (P 7626), sequencing grade trypsin (T 6567), and anti-band 3 N-terminus monoclonal antibody (B 9277) were purchased from Sigma-Aldrich Inc, USA.

    Techniques: In Vitro, Incubation, Western Blot, Staining

    Enhanced growth inhibition effect of the vacuolar (H + )‐ ATP ase inhibitor by pretreatment with a cathepsin D inhibitor or  CTSD  knockdown. (a,b )  RCC 4 renal cell carcinoma cells plus vector alone ( RCC 4‐vec) and  RKO  colon cancer cells were treated with the indicated concentrations of pepstatin A. After 1 day, these cells were treated with the indicated concentrations of bafilomycin A1. After 3 days, cell viability was assessed. Ordinate values were obtained by setting the control group value as 100%. Data are presented as mean ±  SD  ( n  = 3). * P

    Journal: Cancer Science

    Article Title: Cancer with low cathepsin D levels is susceptible to vacuolar (H+)‐ATPase inhibition

    doi: 10.1111/cas.13240

    Figure Lengend Snippet: Enhanced growth inhibition effect of the vacuolar (H + )‐ ATP ase inhibitor by pretreatment with a cathepsin D inhibitor or CTSD knockdown. (a,b ) RCC 4 renal cell carcinoma cells plus vector alone ( RCC 4‐vec) and RKO colon cancer cells were treated with the indicated concentrations of pepstatin A. After 1 day, these cells were treated with the indicated concentrations of bafilomycin A1. After 3 days, cell viability was assessed. Ordinate values were obtained by setting the control group value as 100%. Data are presented as mean ± SD ( n = 3). * P

    Article Snippet: Either 30 μM or 100 μM pepstatin A (LKT Laboratories, St. Paul, MN, USA), 10 μM E‐64‐d (Peptide Institute, Osaka, Japan), 10 μM CA‐074 (Peptide Institute), 10 μM cathepsin K inhibitor II (Merck Millipore, Darmstadt, Germany), 30 μM CAA0225 (Merck Millipore), or 10 μM cathepsin L inhibitor III (Merck Millipore) was added 24 h post‐seeding, and indicated concentrations of bafilomycin A1 were added after 24 h. After 3 days, cell viability was assessed by a Cell Titer‐Glo Luminescent Cell Viability Assay (Promega).

    Techniques: Inhibition, Plasmid Preparation

    Amino acid sequence encoded by exon 4 determines APOL1 cytotoxicity. A : fluorescence microscopy images of HEK293T cells transfected with a control vector (pcDNA3.1) or vectors expressing APOL1 splice variants or deletion mutants and stained 24 h later with the DNA dyes Hoechst 33342 (blue) and propidium iodide (PI; red). Scale bars = 100 μm. B : HEK293T cells were transfected in triplicates with APOL1 expression vectors or control pcDNA3.1 (solid bars) and cultured in medium supplemented with 10% FCS. To analyze whether lysosomal damage contributes to cytotoxicity, at 5 h after transfection the culture medium was replaced with medium supplemented with 2% FCS, and cells were treated with the lysosomal protease inhibitors E64d and pepstatin A (denoted E64d) (hatched bars). At 24 h after transfection, cell lysates and culture supernatants were collected. Culture supernatants were subjected to lactate dehydrogenase (LDH) assay. Cytotoxicity of APOL1 variants is expressed relative to completely lysed control cells, which is set as 100%. Values are means ± SD of 3 independent samples. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Exon 4-encoded sequence is a major determinant of cytotoxicity of apolipoprotein L1

    doi: 10.1152/ajpcell.00384.2014

    Figure Lengend Snippet: Amino acid sequence encoded by exon 4 determines APOL1 cytotoxicity. A : fluorescence microscopy images of HEK293T cells transfected with a control vector (pcDNA3.1) or vectors expressing APOL1 splice variants or deletion mutants and stained 24 h later with the DNA dyes Hoechst 33342 (blue) and propidium iodide (PI; red). Scale bars = 100 μm. B : HEK293T cells were transfected in triplicates with APOL1 expression vectors or control pcDNA3.1 (solid bars) and cultured in medium supplemented with 10% FCS. To analyze whether lysosomal damage contributes to cytotoxicity, at 5 h after transfection the culture medium was replaced with medium supplemented with 2% FCS, and cells were treated with the lysosomal protease inhibitors E64d and pepstatin A (denoted E64d) (hatched bars). At 24 h after transfection, cell lysates and culture supernatants were collected. Culture supernatants were subjected to lactate dehydrogenase (LDH) assay. Cytotoxicity of APOL1 variants is expressed relative to completely lysed control cells, which is set as 100%. Values are means ± SD of 3 independent samples. * P

    Article Snippet: Where indicated, culture media were supplemented with a mixture of lysosomal protease inhibitors and E64d and pepstatin A (both at 10 μg/ml; Tocris Bioscience).

    Techniques: Sequencing, Fluorescence, Microscopy, Transfection, Plasmid Preparation, Expressing, Staining, Cell Culture, Lactate Dehydrogenase Assay

    Expression of IL-8 after pepsin exposure. a Change in inflammatory cytokine expression of Hep-2 and Tu212 cells exposed to different concentrations of pepsin measured using the CBA assay. Production of IL-8 and IL-6 by Hep-2 and Tu212 cells at different pepsin concentrations. b Change in inflammatory cytokine expression of Hep-2 and Tu212 cells exposed to pepsin with/without pepstatin measured using CBA assays. Production of IL-8 and IL-6 by Hep-2 and Tu212 cells at different concentrations of pepsin. c Photomicrographs representative of the immunohistochemical analyses of pepsin and IL-8 in tissue specimens from three patients with laryngeal carcinoma (magnification, ×400). * P

    Journal: Cancer Cell International

    Article Title: Pepsin promotes IL-8 signaling-induced epithelial–mesenchymal transition in laryngeal carcinoma

    doi: 10.1186/s12935-019-0772-7

    Figure Lengend Snippet: Expression of IL-8 after pepsin exposure. a Change in inflammatory cytokine expression of Hep-2 and Tu212 cells exposed to different concentrations of pepsin measured using the CBA assay. Production of IL-8 and IL-6 by Hep-2 and Tu212 cells at different pepsin concentrations. b Change in inflammatory cytokine expression of Hep-2 and Tu212 cells exposed to pepsin with/without pepstatin measured using CBA assays. Production of IL-8 and IL-6 by Hep-2 and Tu212 cells at different concentrations of pepsin. c Photomicrographs representative of the immunohistochemical analyses of pepsin and IL-8 in tissue specimens from three patients with laryngeal carcinoma (magnification, ×400). * P

    Article Snippet: The pepsin inhibitor pepstatin A and the interleukin-8 (IL-8) inhibitor SB225002 were synthetized by Selleckchem (Shanghai, China).

    Techniques: Expressing, Crocin Bleaching Assay, Immunohistochemistry

    Expression of epithelial and mesenchymal markers in Hep-2 and Tu212 cells treated with pepsin. a Morphology of Hep-2 and Tu212 cells exposed to different pepsin concentrations is shown using phase contrast microscopy. b Expression levels of E-cadherin, vimentin, and β-catenin in Hep-2 and Tu212 cells exposed to different concentrations of pepsin analyzed using quantitative real-time PCR. c Effect of different pepsin concentrations on the expression of E-cadherin and vimentin in Hep-2 and Tu212 cells immunostained and analyzed using confocal microscopy (magnification, ×200). d Expression of E-cadherin, vimentin, β-catenin, snail, and slug in Hep-2 and Tu212 cells exposed to different concentrations of pepsin analyzed using western blotting. e Expression of E-cadherin, vimentin, β-catenin, snail, and slug in Hep-2 and Tu212 cells exposed to pepsin with/without pepstatin analyzed using western blotting. f Representative photomicrographs illustrating the immunohistochemical analyses for pepsin, E-cadherin, vimentin, and β-catenin in tissue specimens from two patients with laryngeal carcinoma (magnification, ×400). * P

    Journal: Cancer Cell International

    Article Title: Pepsin promotes IL-8 signaling-induced epithelial–mesenchymal transition in laryngeal carcinoma

    doi: 10.1186/s12935-019-0772-7

    Figure Lengend Snippet: Expression of epithelial and mesenchymal markers in Hep-2 and Tu212 cells treated with pepsin. a Morphology of Hep-2 and Tu212 cells exposed to different pepsin concentrations is shown using phase contrast microscopy. b Expression levels of E-cadherin, vimentin, and β-catenin in Hep-2 and Tu212 cells exposed to different concentrations of pepsin analyzed using quantitative real-time PCR. c Effect of different pepsin concentrations on the expression of E-cadherin and vimentin in Hep-2 and Tu212 cells immunostained and analyzed using confocal microscopy (magnification, ×200). d Expression of E-cadherin, vimentin, β-catenin, snail, and slug in Hep-2 and Tu212 cells exposed to different concentrations of pepsin analyzed using western blotting. e Expression of E-cadherin, vimentin, β-catenin, snail, and slug in Hep-2 and Tu212 cells exposed to pepsin with/without pepstatin analyzed using western blotting. f Representative photomicrographs illustrating the immunohistochemical analyses for pepsin, E-cadherin, vimentin, and β-catenin in tissue specimens from two patients with laryngeal carcinoma (magnification, ×400). * P

    Article Snippet: The pepsin inhibitor pepstatin A and the interleukin-8 (IL-8) inhibitor SB225002 were synthetized by Selleckchem (Shanghai, China).

    Techniques: Expressing, Microscopy, Real-time Polymerase Chain Reaction, Confocal Microscopy, Western Blot, Immunohistochemistry

    Impact of Pepstatin A on neutrophil migration induced by patient gastric fluid samples set at pH 3 and pH 7.4. Pepstatin A (20 µg/ml) was added to gastric samples set at pH 3 and pH 7.4. Pepstatin A failed to significantly reduce the magnitude of neutrophil (PMN) migration across H292 lung epithelium induced by gastric samples set at pH 3 from patients off PPI therapy ( A ), but did significantly block neutrophil migration induced by gastric samples set at pH 3 from patients on PPI therapy ( B ). For gastric fluid samples set at pH 7.4, Pepstatin A failed to impact the magnitude of neutrophil migration induced by samples collected from patients off ( C ) and on ( D ) PPI therapy. A dilution of 1:4 in pH matched HBSS was used for all samples. Both on and off PPI therapy groups set at either pH 3 or 7.4 represent the mean +/− SEM of the average magnitude values (n = 3) of PMN migration by gastric fluid derived from 12 patients on PPI therapy and 12 patients off PPI therapy, diluted 1:4 in HBSS and treated with or without Pepstatin A (n = 12). Differences were considered significant between groups with and without Pepstatin A at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. Panels A–D are representative internally controlled experiments conducted on at least two separate occasions yielding similar results.

    Journal: Scientific Reports

    Article Title: Pepsin Triggers Neutrophil Migration Across Acid Damaged Lung Epithelium

    doi: 10.1038/s41598-019-50360-4

    Figure Lengend Snippet: Impact of Pepstatin A on neutrophil migration induced by patient gastric fluid samples set at pH 3 and pH 7.4. Pepstatin A (20 µg/ml) was added to gastric samples set at pH 3 and pH 7.4. Pepstatin A failed to significantly reduce the magnitude of neutrophil (PMN) migration across H292 lung epithelium induced by gastric samples set at pH 3 from patients off PPI therapy ( A ), but did significantly block neutrophil migration induced by gastric samples set at pH 3 from patients on PPI therapy ( B ). For gastric fluid samples set at pH 7.4, Pepstatin A failed to impact the magnitude of neutrophil migration induced by samples collected from patients off ( C ) and on ( D ) PPI therapy. A dilution of 1:4 in pH matched HBSS was used for all samples. Both on and off PPI therapy groups set at either pH 3 or 7.4 represent the mean +/− SEM of the average magnitude values (n = 3) of PMN migration by gastric fluid derived from 12 patients on PPI therapy and 12 patients off PPI therapy, diluted 1:4 in HBSS and treated with or without Pepstatin A (n = 12). Differences were considered significant between groups with and without Pepstatin A at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. Panels A–D are representative internally controlled experiments conducted on at least two separate occasions yielding similar results.

    Article Snippet: Treatment of airway cells with Pepstatin A Pepstatin A (Sigma-Aldrich, St. Louis, MO) was resuspended in DMSO at 10 mg/ml, aliquoted, and stored at −20 °C.

    Techniques: Migration, Blocking Assay, Derivative Assay, Two Tailed Test

    Evaluation of the impact of gastric fluid samples and pepsin on neutrophil migration using a human basal stem cell derived airway mucosa model cultured at air-liquid interface. Inverted air-liquid interface (ALI) airway mucosa cultured on inverted 3.0 μm pore sized permeable transwell filters were wholemount stained to highlight ciliated cells (AcTub Ezrin), tight junctions (ZO-1), and nuclei (DAPI). Scale bar, 20 μm for ZO-1 field, 50 μm for AcTub Ezrin ( A ). Pepsin (29 µg/ml) set at pH 3 and a gastric fluid sample set at pH 3 and diluted 1:4 in pH matched HBSS derived from a patient on PPI therapy both induce a significant magnitude of neutrophil (PMN) migration across a human primary airway mucosa grown at air-liquid interface ( B ). HBSS set at pH 3 and a gastric fluid sample derived from the patient off PPI therapy set at pH 3 failed to induce neutrophil migration as did all conditions set at pH 7.4 ( B ). These effects observed with pepsin and the gastric sample derived from a patient on PPI therapy, both set at pH 3, are completely abrogated by the addition of Pepstatin A (20 µg/ml) ( C ). Panels A and B are separate internally controlled experiments. Each data point depicts the mean +/− SD of triplicate wells assayed (n = 3). Differences were considered significant at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. The gastric sample from the patient on PPI therapy was patient #18 and the gastric sample from the patient off PPI therapy was patient #11 (Supplementary Table S1 ).

    Journal: Scientific Reports

    Article Title: Pepsin Triggers Neutrophil Migration Across Acid Damaged Lung Epithelium

    doi: 10.1038/s41598-019-50360-4

    Figure Lengend Snippet: Evaluation of the impact of gastric fluid samples and pepsin on neutrophil migration using a human basal stem cell derived airway mucosa model cultured at air-liquid interface. Inverted air-liquid interface (ALI) airway mucosa cultured on inverted 3.0 μm pore sized permeable transwell filters were wholemount stained to highlight ciliated cells (AcTub Ezrin), tight junctions (ZO-1), and nuclei (DAPI). Scale bar, 20 μm for ZO-1 field, 50 μm for AcTub Ezrin ( A ). Pepsin (29 µg/ml) set at pH 3 and a gastric fluid sample set at pH 3 and diluted 1:4 in pH matched HBSS derived from a patient on PPI therapy both induce a significant magnitude of neutrophil (PMN) migration across a human primary airway mucosa grown at air-liquid interface ( B ). HBSS set at pH 3 and a gastric fluid sample derived from the patient off PPI therapy set at pH 3 failed to induce neutrophil migration as did all conditions set at pH 7.4 ( B ). These effects observed with pepsin and the gastric sample derived from a patient on PPI therapy, both set at pH 3, are completely abrogated by the addition of Pepstatin A (20 µg/ml) ( C ). Panels A and B are separate internally controlled experiments. Each data point depicts the mean +/− SD of triplicate wells assayed (n = 3). Differences were considered significant at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. The gastric sample from the patient on PPI therapy was patient #18 and the gastric sample from the patient off PPI therapy was patient #11 (Supplementary Table S1 ).

    Article Snippet: Treatment of airway cells with Pepstatin A Pepstatin A (Sigma-Aldrich, St. Louis, MO) was resuspended in DMSO at 10 mg/ml, aliquoted, and stored at −20 °C.

    Techniques: Migration, Derivative Assay, Cell Culture, Staining, Two Tailed Test

    Mechanisms of pepsin-induced neutrophil migration and barrier integrity disruption. Pepstatin A (20 µg/ml), an inhibitor of pepsin, blocked neutrophil (PMN) migration induced by exposure to pepsin (50 µg/ml) at pH 3 ( A ) and prevented pepsin-induced barrier integrity disruption (HRP flux) at doses of pepsin ranging from 12.5–50 µg/ml pepsin at pH 3 ( B ). Pepstatin A failed to prevent neutrophil migration induced by PAO1 infection or following the application of a chemoattractant gradient of fMLP (100 nM) ( C ). An inhibitor of 12-lipooxygenase (CDC) failed to interfere with neutrophil migration induced by pepsin at pH 3 while PMN migration in response to PAO1 infection at pH 7.4 was significantly reduced ( D ). Exposure of pepsin (50 µg/ml) to NaOH bringing the solution pH up to between 8.5 and 10 followed by return to pH 3 by adding HCl and then applying to H292 lung epithelial monolayers abolished both the PMN transmigratory response and the increased HRP flux associated with control pepsin (50 µg/ml) at pH 3 treated in parallel with HCl only ( E ). Panels A–E are representative internally controlled experiments conducted on at least three separate occasions yielding similar results. Each data point depicts the mean +/− SD of triplicate wells assayed (n = 3). Differences were considered significant at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment.

    Journal: Scientific Reports

    Article Title: Pepsin Triggers Neutrophil Migration Across Acid Damaged Lung Epithelium

    doi: 10.1038/s41598-019-50360-4

    Figure Lengend Snippet: Mechanisms of pepsin-induced neutrophil migration and barrier integrity disruption. Pepstatin A (20 µg/ml), an inhibitor of pepsin, blocked neutrophil (PMN) migration induced by exposure to pepsin (50 µg/ml) at pH 3 ( A ) and prevented pepsin-induced barrier integrity disruption (HRP flux) at doses of pepsin ranging from 12.5–50 µg/ml pepsin at pH 3 ( B ). Pepstatin A failed to prevent neutrophil migration induced by PAO1 infection or following the application of a chemoattractant gradient of fMLP (100 nM) ( C ). An inhibitor of 12-lipooxygenase (CDC) failed to interfere with neutrophil migration induced by pepsin at pH 3 while PMN migration in response to PAO1 infection at pH 7.4 was significantly reduced ( D ). Exposure of pepsin (50 µg/ml) to NaOH bringing the solution pH up to between 8.5 and 10 followed by return to pH 3 by adding HCl and then applying to H292 lung epithelial monolayers abolished both the PMN transmigratory response and the increased HRP flux associated with control pepsin (50 µg/ml) at pH 3 treated in parallel with HCl only ( E ). Panels A–E are representative internally controlled experiments conducted on at least three separate occasions yielding similar results. Each data point depicts the mean +/− SD of triplicate wells assayed (n = 3). Differences were considered significant at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment.

    Article Snippet: Treatment of airway cells with Pepstatin A Pepstatin A (Sigma-Aldrich, St. Louis, MO) was resuspended in DMSO at 10 mg/ml, aliquoted, and stored at −20 °C.

    Techniques: Migration, Infection, Two Tailed Test

    Abrogation of CathD activity leads to increased Caspase-3 activity in MCF-7 cells after the induction of apoptosis. ( a ) Inhibition of CathD activity by treatment of MCF-7 cells with 40 μ M pepstatin A leads to increased Caspase-3 activity

    Journal: Cell Death and Differentiation

    Article Title: The Apaf-1-binding protein Aven is cleaved by Cathepsin D to unleash its anti-apoptotic potential

    doi: 10.1038/cdd.2012.17

    Figure Lengend Snippet: Abrogation of CathD activity leads to increased Caspase-3 activity in MCF-7 cells after the induction of apoptosis. ( a ) Inhibition of CathD activity by treatment of MCF-7 cells with 40 μ M pepstatin A leads to increased Caspase-3 activity

    Article Snippet: Protease inhibitors pepstatin A (Sigma Aldrich), E-64 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), z-VAD (Sigma), aprotinin and pAPMSF (both Santa Cruz Biotechnology) were dissolved in DMSO (Roth, Karlsruhe, Germany) and used at the concentrations indicated in the text.

    Techniques: Activity Assay, Inhibition

    Western blot of sLOX-1 released from cells treated with MβCD and different protease inhibitors. LOX-1-V5-COS transfected cells were incubated with MβCD, GM6001, PMSF, Pepstatin A and the protease inhibitor cocktail (PI mix) (Calbiochem), as indicated (upper panel). Histogram shows the densitometric analysis expressed as percentage of reduction of sLOX-1 released in the presence of different protease inhibitors. 100% refers as sLOX-1 released in the presence of 5mM MβCD for 30 min (lower panel). Data in histograms represent the average ± SEM of three experiments, P

    Journal: PLoS ONE

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

    doi: 10.1371/journal.pone.0141270

    Figure Lengend Snippet: Western blot of sLOX-1 released from cells treated with MβCD and different protease inhibitors. LOX-1-V5-COS transfected cells were incubated with MβCD, GM6001, PMSF, Pepstatin A and the protease inhibitor cocktail (PI mix) (Calbiochem), as indicated (upper panel). Histogram shows the densitometric analysis expressed as percentage of reduction of sLOX-1 released in the presence of different protease inhibitors. 100% refers as sLOX-1 released in the presence of 5mM MβCD for 30 min (lower panel). Data in histograms represent the average ± SEM of three experiments, P

    Article Snippet: Phenylmethylsulfonylfuoride (PMSF) (Euroclone, Devon, UK), pepstatin A (Boehringer, Mannheim, DE), GM6001 inhibitor (Biomol Research Lab Inc.), protease inhibitor cocktail set III (AEBSF 100mM; aprotinin 80μM; bestatin 5mM; E-64 1,5mM; leupeptin 2mM; pepstatin A 1mM) (Calbiochem, La Jolla, CA) were used for specific protease class inhibition experiments.

    Techniques: Western Blot, Transfection, Incubation, Protease Inhibitor

    EA.hy926 cells secretome induce LOX-1 shedding. (A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with DMEM (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).

    Journal: PLoS ONE

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

    doi: 10.1371/journal.pone.0141270

    Figure Lengend Snippet: EA.hy926 cells secretome induce LOX-1 shedding. (A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with DMEM (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).

    Article Snippet: Phenylmethylsulfonylfuoride (PMSF) (Euroclone, Devon, UK), pepstatin A (Boehringer, Mannheim, DE), GM6001 inhibitor (Biomol Research Lab Inc.), protease inhibitor cocktail set III (AEBSF 100mM; aprotinin 80μM; bestatin 5mM; E-64 1,5mM; leupeptin 2mM; pepstatin A 1mM) (Calbiochem, La Jolla, CA) were used for specific protease class inhibition experiments.

    Techniques: Western Blot, Derivative Assay, Transfection, Incubation

    WNV-NY replication does not induce autophagy in 293T cells. (A) Effect of protease inhibitors on WNV replication. 293T cells infected with WNV-NY (MOI = 3) were incubated in the presence (Prot. Inhibit.) or absence (None) of 10 µg/mL Pepstatin A and E64d. Media was removed from cultures at the indicated times, cleared of cell debris, and titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from two independent experiments. (B) Effect of WNV-NY infection on steady-state levels of LC3B-II. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3). After infection, the inoculum was replaced with medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV-NY infection on steady-state levels of LC3B-II and p62. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), GAPDH (third panel), and WNV (bottom panel). (D) Effect of WNV infection of levels of p62. 293T cells were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of p62 (top), GAPDH (middle) or WNV (bottom).

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: WNV-NY replication does not induce autophagy in 293T cells. (A) Effect of protease inhibitors on WNV replication. 293T cells infected with WNV-NY (MOI = 3) were incubated in the presence (Prot. Inhibit.) or absence (None) of 10 µg/mL Pepstatin A and E64d. Media was removed from cultures at the indicated times, cleared of cell debris, and titers determined by plaque assay on Vero cells. Values represent the average (± standard error) number of plaque-forming units (pfu) per mL from two independent experiments. (B) Effect of WNV-NY infection on steady-state levels of LC3B-II. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3). After infection, the inoculum was replaced with medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV-NY infection on steady-state levels of LC3B-II and p62. 293T monolayers were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), GAPDH (third panel), and WNV (bottom panel). (D) Effect of WNV infection of levels of p62. 293T cells were mock-infected or infected with WNV-NY (MOI = 3) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of p62 (top), GAPDH (middle) or WNV (bottom).

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection, Incubation, Plaque Assay, Expressing

    LC3B-II accumulation in 293T cells. (A) Effect of WNV infection on LC3-II levels. Whole cell lysates were prepared from mock-infected or WNV-infected (MOI = 3) 293T cells at 24 hours post-infection. Immunoblot anyalysis was used to determine steady-state levels of LC3B (top), GAPDH (middle), and WNV (bottom). (B) Effect of rapamycin on LC3B. Whole cell lysates prepared at the indicated times (h) from 293T cells treated with 100 nM rapamycin (rap) were subjected to immunoblot analysis to determine the steady-state levels of LC3B (top) and GAPDH (bottom). (C) Effect of protease inhibitors on LC3B-II levels. Whole cell lysates were prepared from 293T monolayers treated with rapamycin (Rap) in the presence or absence of pepstatin A (Pep) and/or E64d at 24 hours post-treatment. Lysates were subjected to immunoblot analysis as described in Panel B.

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: LC3B-II accumulation in 293T cells. (A) Effect of WNV infection on LC3-II levels. Whole cell lysates were prepared from mock-infected or WNV-infected (MOI = 3) 293T cells at 24 hours post-infection. Immunoblot anyalysis was used to determine steady-state levels of LC3B (top), GAPDH (middle), and WNV (bottom). (B) Effect of rapamycin on LC3B. Whole cell lysates prepared at the indicated times (h) from 293T cells treated with 100 nM rapamycin (rap) were subjected to immunoblot analysis to determine the steady-state levels of LC3B (top) and GAPDH (bottom). (C) Effect of protease inhibitors on LC3B-II levels. Whole cell lysates were prepared from 293T monolayers treated with rapamycin (Rap) in the presence or absence of pepstatin A (Pep) and/or E64d at 24 hours post-treatment. Lysates were subjected to immunoblot analysis as described in Panel B.

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection

    The autophagy pathway is not upregulated by WNV in established cell lines. (A and B) Effect of WNV infection on steady-state LC3B-II levels in Huh7 (A) or Huh7.5 (B) cells. Following mock or WNV-NY (MOI = 3) infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV and SINV infection of steady-state LC3B-II and p62 levels in Neuro2A cells. Neuro2A cells were mock-infected or infected with WNV (MOI = 3) or SINV (MOI = 5) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), WNV (third panel), and SINV (bottom panel).

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: The autophagy pathway is not upregulated by WNV in established cell lines. (A and B) Effect of WNV infection on steady-state LC3B-II levels in Huh7 (A) or Huh7.5 (B) cells. Following mock or WNV-NY (MOI = 3) infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle), and WNV (bottom). (C) Effect of WNV and SINV infection of steady-state LC3B-II and p62 levels in Neuro2A cells. Neuro2A cells were mock-infected or infected with WNV (MOI = 3) or SINV (MOI = 5) in the absence of protease inhibitors. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top panel), p62 (second panel), WNV (third panel), and SINV (bottom panel).

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection, Expressing

    WNV-MAD78 infection does not induce autophagy. 293T monolayers were mock-infected or infected with WNV-MAD78 (MOI = 3). Following infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for LC3B (top), GAPDH (middle) and WNV (bottom).

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: WNV-MAD78 infection does not induce autophagy. 293T monolayers were mock-infected or infected with WNV-MAD78 (MOI = 3). Following infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for LC3B (top), GAPDH (middle) and WNV (bottom).

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection

    Primary human cell lines do not upregulate autophagy in response to WNV infection. (A-B) Effect of WNV infection on steady-state LC3B-II levels. Human foreskin fibroblasts (A) or human brain cortical astrocytes (B) were mock-infected or infected with WNV-NY (MOI = 3). Following infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle) and WNV (bottom).

    Journal: PLoS ONE

    Article Title: West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

    doi: 10.1371/journal.pone.0045800

    Figure Lengend Snippet: Primary human cell lines do not upregulate autophagy in response to WNV infection. (A-B) Effect of WNV infection on steady-state LC3B-II levels. Human foreskin fibroblasts (A) or human brain cortical astrocytes (B) were mock-infected or infected with WNV-NY (MOI = 3). Following infection, cells were grown in medium containing 10 µg/mL Pepstatin A and E64d. Whole cell lysates prepared at the indicated times (h) post-infection were subjected to immunoblot analysis for expression of LC3B (top), GAPDH (middle) and WNV (bottom).

    Article Snippet: Small Molecules The protease inhibitors E64d (Sigma) and Pepstatin A (Fisher Scientific) were dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 10 µg/mL in complete DMEM.

    Techniques: Infection, Expressing

    Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10 8 ) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.

    Article Snippet: These results indicate that aspartate proteases also contribute to degradation of the Ii chain in addition to cysteine proteases, such as cathepsin S. Thus, subjecting mice to pepstatin A may have impaired OVA-specific proliferation and activation of CD4+ T cells by suppressing maturation of the MHC class II molecule and antigen presentation.

    Techniques: Mouse Assay, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Silver Staining

    In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

    Article Snippet: These results indicate that aspartate proteases also contribute to degradation of the Ii chain in addition to cysteine proteases, such as cathepsin S. Thus, subjecting mice to pepstatin A may have impaired OVA-specific proliferation and activation of CD4+ T cells by suppressing maturation of the MHC class II molecule and antigen presentation.

    Techniques: In Vitro, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

    T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10 6 cells. Standard deviations (SD) were

    Article Snippet: These results indicate that aspartate proteases also contribute to degradation of the Ii chain in addition to cysteine proteases, such as cathepsin S. Thus, subjecting mice to pepstatin A may have impaired OVA-specific proliferation and activation of CD4+ T cells by suppressing maturation of the MHC class II molecule and antigen presentation.

    Techniques: Mouse Assay, Injection, Enzyme-linked Immunospot

    The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n  = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice ( n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

    Article Snippet: These results indicate that aspartate proteases also contribute to degradation of the Ii chain in addition to cysteine proteases, such as cathepsin S. Thus, subjecting mice to pepstatin A may have impaired OVA-specific proliferation and activation of CD4+ T cells by suppressing maturation of the MHC class II molecule and antigen presentation.

    Techniques: Mouse Assay

    Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n  = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

    Journal: Immunology

    Article Title: Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

    doi: 10.1046/j.1365-2567.2000.00000.x

    Figure Lengend Snippet: Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals ( n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

    Article Snippet: These results indicate that aspartate proteases also contribute to degradation of the Ii chain in addition to cysteine proteases, such as cathepsin S. Thus, subjecting mice to pepstatin A may have impaired OVA-specific proliferation and activation of CD4+ T cells by suppressing maturation of the MHC class II molecule and antigen presentation.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Ex vivo invasive activity of VSMCs in 3-D collagen. (a–c) Media explants of mouse aorta were embedded within a 3-D gel of type I collagen, VSMC egress was initiated by exogenous PDGF/FGF-2, and outgrowth into the translucent collagen matrix was visualized by darkfield microscopy at 0, 3, and 8 d. Asterisk indicates explant , and arrowheads point at the leading edge of the invading SMC. Bar, 1 mm. Inset in (a) shows anti-SM α-actin staining (brown) of the media explant. (d and e) Scanning electron micrograph of freeze-fractured explant cultures. Asterisks mark invading VSMCs that have infiltrated into the field of densely packed type I collagen fibrils. Arrows highlight VSMC cytoplasmic extensions. Tunnels (star) in the collagen matrix are seen in areas surrounding the embedded explant. Bar, 20 μm. (f) Degraded collagen (detected by mAb HUI77) appears as punctate green staining (arrows) surrounding propidium iodide–labeled cells (red, asterisks). Bar, 20 μm. (g–o) Media explants from WT, plasminogen (plg)-, MMP-9–, or MMP-2–null mice were suspended in collagen in autologous sera and cultured for 8 d in the absence or presence of E-64, pepstatin, aprotinin, BB-94, TIMP-1, or TIMP-2 as described in Materials and methods. Insets in j, k, n, and o show immunostains for degraded collagen (mAb HUI77) surrounding propidium iodide–labeled cells. Bar, 1 mm. (p) Quantitative analysis of VSMC invasive activity in aortic explant cultures after an 8-d culture period. Results are expressed as the mean ± SEM ( n = 5).

    Journal: The Journal of Experimental Medicine

    Article Title: MT1-matrix metalloproteinase directs arterial wall invasion and neointima formation by vascular smooth muscle cells

    doi: 10.1084/jem.20050607

    Figure Lengend Snippet: Ex vivo invasive activity of VSMCs in 3-D collagen. (a–c) Media explants of mouse aorta were embedded within a 3-D gel of type I collagen, VSMC egress was initiated by exogenous PDGF/FGF-2, and outgrowth into the translucent collagen matrix was visualized by darkfield microscopy at 0, 3, and 8 d. Asterisk indicates explant , and arrowheads point at the leading edge of the invading SMC. Bar, 1 mm. Inset in (a) shows anti-SM α-actin staining (brown) of the media explant. (d and e) Scanning electron micrograph of freeze-fractured explant cultures. Asterisks mark invading VSMCs that have infiltrated into the field of densely packed type I collagen fibrils. Arrows highlight VSMC cytoplasmic extensions. Tunnels (star) in the collagen matrix are seen in areas surrounding the embedded explant. Bar, 20 μm. (f) Degraded collagen (detected by mAb HUI77) appears as punctate green staining (arrows) surrounding propidium iodide–labeled cells (red, asterisks). Bar, 20 μm. (g–o) Media explants from WT, plasminogen (plg)-, MMP-9–, or MMP-2–null mice were suspended in collagen in autologous sera and cultured for 8 d in the absence or presence of E-64, pepstatin, aprotinin, BB-94, TIMP-1, or TIMP-2 as described in Materials and methods. Insets in j, k, n, and o show immunostains for degraded collagen (mAb HUI77) surrounding propidium iodide–labeled cells. Bar, 1 mm. (p) Quantitative analysis of VSMC invasive activity in aortic explant cultures after an 8-d culture period. Results are expressed as the mean ± SEM ( n = 5).

    Article Snippet: Where indicated, protease inhibitors were added to media explants or isolated VSMCs at the following final concentrations: 3 μM BB-94 (in 0.1% DMSO final; gift of British Biotechnology Ltd.), 5 μg/ml TIMP-2, 12.5 μg/ml TIMP-1 (equimolar as determined by active site titration [ ]; endotoxin-free; Fuji Industries Co., Ltd.), 100 μM E-64 (in 0.05% ethanol final, Sigma-Aldrich), 100 μg/ml aprotinin (Roche), or 50 μM pepstatin (in 0.05% ethanol final; Roche).

    Techniques: Ex Vivo, Activity Assay, Microscopy, Staining, Labeling, Mouse Assay, Cell Culture

    Effect of TXA1 on the expression levels of LC3-II in A375-C5 cells. Cells were treated for 48 h with medium (blank), TXA1 (3.6 μM or 7.2 μM), or with the corresponding DMSO concentrations (DMSO or DMSO2, respectively) LC3-II protein levels were analyzed by Western blot. ( A ) Following treatment with TXA1 alone; ( B ) following co-treatment with 3-MA; and ( C ) following co-treatment with E-64d/pepstatin. Images are representative of, at least, three independent experiments (except for the case of blank and DMSO treatments in the presence of E-64d/pepstatin, which result from two experiments only). Results of the densitometry analysis are expressed after normalization of the values obtained for each protein with the values obtained for tubulin or actin (and further expressed in relation to blank cells) and represent the mean ± SEM from, at least, three independent experiments (except for the case of blank and DMSO treatments in the presence of E-64d/pepstatin, which result from two experiments only). * p ≤ 0.05 Blank vs. treatment.

    Journal: Molecules

    Article Title: Modulation of Autophagy by a Thioxanthone Decreases the Viability of Melanoma Cells

    doi: 10.3390/molecules21101343

    Figure Lengend Snippet: Effect of TXA1 on the expression levels of LC3-II in A375-C5 cells. Cells were treated for 48 h with medium (blank), TXA1 (3.6 μM or 7.2 μM), or with the corresponding DMSO concentrations (DMSO or DMSO2, respectively) LC3-II protein levels were analyzed by Western blot. ( A ) Following treatment with TXA1 alone; ( B ) following co-treatment with 3-MA; and ( C ) following co-treatment with E-64d/pepstatin. Images are representative of, at least, three independent experiments (except for the case of blank and DMSO treatments in the presence of E-64d/pepstatin, which result from two experiments only). Results of the densitometry analysis are expressed after normalization of the values obtained for each protein with the values obtained for tubulin or actin (and further expressed in relation to blank cells) and represent the mean ± SEM from, at least, three independent experiments (except for the case of blank and DMSO treatments in the presence of E-64d/pepstatin, which result from two experiments only). * p ≤ 0.05 Blank vs. treatment.

    Article Snippet: Treatment with Autophagy Inhibitors Cells were plated in six-well plates (1 × 105 cells/well) and allowed to adhere for 24 h. Cells were then treated for 1 h with 0.5 mM 3-methyladenine (3-MA, Sigma) or with 10 μg/mL E-64d (AppliChem, Darmstadt, Germany) and Pepstatin A (Cayman Chemical, Ann Arbor, MI, USA) and then co-incubated with 3.6 μM of TXA1, or with control treatments (blank, DMSO) for 48 h. Viable cell numbers were then assessed by trypan blue assay and protein expression analyzed by Western blot, as described above.

    Techniques: Expressing, Western Blot