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  • 99
    Thermo Fisher pepstatin a
    Pepstatin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pepstatin a
    Pepstatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10356 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
    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
    Santa Cruz Biotechnology pepstatin a
    PES-induced cell death, BID processing and caspase 8 cleavage are reduced by pepstatin A. ( a ) BC3 and BCBL1 mock-treated or treated with PES (20 μ M for 24 h) in the presence or absence of <t>pepstatin</t> A (20 μ M). Cells were counted by trypan-blue exclusion and the mean±S.D. is reported * P -value=0.02, ** P -value=0.02. ( b ) Western blot was also performed concomitantly with the treatments reported above to analyze the processing of Bid and caspase 8. β -Actin was used as loading control and a representative experiment out of three is shown
    Pepstatin A, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pepstatina
    PES-induced cell death, BID processing and caspase 8 cleavage are reduced by pepstatin A. ( a ) BC3 and BCBL1 mock-treated or treated with PES (20 μ M for 24 h) in the presence or absence of <t>pepstatin</t> A (20 μ M). Cells were counted by trypan-blue exclusion and the mean±S.D. is reported * P -value=0.02, ** P -value=0.02. ( b ) Western blot was also performed concomitantly with the treatments reported above to analyze the processing of Bid and caspase 8. β -Actin was used as loading control and a representative experiment out of three is shown
    Pepstatina, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15781 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem pepstatin a
    The effect of cathepsin D inhibition on the degradation and activity of extracellular Cys-C and cysteine cathepsins, respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or <t>pepstatin</t> A ( 4 ), grassystatins D and F ( 1 and 3 ) for 3 days at 37 ºC. A, B) The concentration of Cys-C in the 3-day CM was quantified by human Cys-C ELISA. C, D) The activity of cysteine cathepsins was quantified by incubating the 3-day CM in assay buffer (37 °C, pH 5.5) in the presence of fluorogenic substrate. The asterisks denote significance of P
    Pepstatin A, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Applichem pepstatin a
    The effect of cathepsin D inhibition on the degradation and activity of extracellular Cys-C and cysteine cathepsins, respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or <t>pepstatin</t> A ( 4 ), grassystatins D and F ( 1 and 3 ) for 3 days at 37 ºC. A, B) The concentration of Cys-C in the 3-day CM was quantified by human Cys-C ELISA. C, D) The activity of cysteine cathepsins was quantified by incubating the 3-day CM in assay buffer (37 °C, pH 5.5) in the presence of fluorogenic substrate. The asterisks denote significance of P
    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
    Bio-Rad pepstatin a
    The effect of cathepsin D inhibition on the degradation and activity of extracellular Cys-C and cysteine cathepsins, respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or <t>pepstatin</t> A ( 4 ), grassystatins D and F ( 1 and 3 ) for 3 days at 37 ºC. A, B) The concentration of Cys-C in the 3-day CM was quantified by human Cys-C ELISA. C, D) The activity of cysteine cathepsins was quantified by incubating the 3-day CM in assay buffer (37 °C, pH 5.5) in the presence of fluorogenic substrate. The asterisks denote significance of P
    Pepstatin A, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 53 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
    Amresco pepstatin a
    CIT induced autophagy-dependent apoptosis in HepG2 cells. Caspase-3 activity was determined using colorimetric method and was expressed as a percent of control activity. ( A ) HepG2 cells were treated with 5 µM CIT for 6 h, 12 h, or 24 h. ( B ) HepG2 cells were transfected with either 50 nM siRNA against human Atg5 (Si Atg5) or scrambled control siRNA (Si Control), and then treated with 5 µM CIT for 24 h. ( C ) HepG2 cells were pretreated with 40 µM <t>pepstatin</t> A for 4 h, and subsequently treated with 5 µM CIT for 24 h. ** p
    Pepstatin A, supplied by Amresco, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher bodipy fl pepstatin a
    CIT induced autophagy-dependent apoptosis in HepG2 cells. Caspase-3 activity was determined using colorimetric method and was expressed as a percent of control activity. ( A ) HepG2 cells were treated with 5 µM CIT for 6 h, 12 h, or 24 h. ( B ) HepG2 cells were transfected with either 50 nM siRNA against human Atg5 (Si Atg5) or scrambled control siRNA (Si Control), and then treated with 5 µM CIT for 24 h. ( C ) HepG2 cells were pretreated with 40 µM <t>pepstatin</t> A for 4 h, and subsequently treated with 5 µM CIT for 24 h. ** p
    Bodipy Fl Pepstatin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bachem pepstatin a
    Cathepsin D expression (a) and activity (b) in the jejunum of control (open bars) and methotrexate (MTX)-treated rats receiving vehicle (closed bars), <t>pepstatin</t> A (dark grey bars) or E64 (grey bars). Values are means ± standard error of the mean
    Pepstatin A, supplied by Bachem, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co pepstatin a
    Effects of P140 on autophagy in U-251 MG glioblastoma cells. U-251 MG cells were treated or not with 10 μM P140 for 8 h in the presence or absence of lysosomal enzyme inhibitors <t>pepstatin</t> A (PepA) and E64D. Protein lysates were separated on 4–20% SDS-gels and then transferred onto nitrocellulose membrane. (A) Proteins were revealed with Abs to ATG12/5, MAP1LC3B, SQSTM1, BECLIN-1, and LAMP2A. Comparisons were made between untreated and P140-treated U-251 MG cells. Normalization was performed by measuring total protein directly on the membrane (stain-free procedure). (B) Autophagic markers MAP1LC3B and SQSTM1 measured in the absence and presence of anti-proteases as indicators of flux. The effect of P140 peptide on the flux intensity is shown. The results are displayed with cells collected after 1–12 cycles of divisions or after a longer period of more than 12–20 cycles of division in culture medium. (C) Effect of P140 on ATG12/5, BECLIN-1, and LAMP2A protein expression. Error bars are SEM. Each sample was tested in triplicates or quadruplets in at least 3 independent experiments (1 point corresponds to the mean value of replicates). P values are from Mann-Whitney U tests.
    Pepstatin A, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bachem acetyl pepstatin
    Calorimetric titrations of wild-type HIV-1 protease with Amprenavir ( left panel), acetyl <t>pepstatin</t> ( center panel), and with Amprenavir in the presence of acetyl pepstatin (displacement titration; right panel). These experiments were performed at 25°C in 10 mM acetate, pH 5, 2% DMSO. In all experiments the protease was in the calorimeter reaction cell and the inhibitors in the injection syringe. The inhibitor was added in stepwise injections of 10 μL. For each experiment the reactant concentrations were: ( left panel) 23.7 μM protease, 250 μM Amprenavir; ( center panel) 18.9 μM protease, 300 μM acetyl pepstatin; ( right panel) 19.1 μM protease, 300 μM acetyl pepstatin, 250 μM Amprenavir.
    Acetyl Pepstatin, supplied by Bachem, used in various techniques. Bioz Stars score: 88/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant pepstatin a
    hnf4 is required for blood feeding a , Graph of the aspartyl protease activity of lysates from control(RNAi) or hnf4(RNAi) parasites as determined by the ability to cleave the fluorogenic substrate, mca-GKPILFFRLK-K(dnp) in the presence of no inhibitor (DMSO), the general cysteine protease inhibitor E-64 (E-64), or the aspartyl protease inhibitor <t>pepstatin</t> A (pepstatin). b , Graph quantifying the recovery rate of worms from transplant recipients. Data are from five recipients. c , Representative photographs of livers of mice 30 days after transplant with RNAi-treated parasites. The number of livers grossly similar to the representative photograph is indicated in the upper right each panel. Data are from two recipients in one biological replicate. d , Graph showing quantification of worm length from Fig. 4f . n = 15 for control(RNAi) male parasites and 16 hnf4(RNAi) male parasites from 3 separate recipients. Scale bar, c , 1cm. ns, not significant, ****, p
    Pepstatin A, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA pepstatin a
    The autophagic flux is increased in DMD myoblast cell lines. ( A – C ) 10 µg of total protein extracts from control (W1 W2) and DMD (D1 to D4) cell lines were separated by SDS-PAGE and analyzed by immunoblots probed with antibodies directed against ( A ) proteins involved in the initiation phase of the autophagic process: PI3K class III, BECN1 and BCL2; ( B ) proteins involved in autophagosome nucleation/elongation or transport/fusion: FOXO3a, ATG3, ATG9L1, ATG7, ATG5/12, HDAC6; ( C ) LC3-I and its lipidated form LC3-II. Actin is used as a loading control. The histograms show means ± SD of the normalized PI3KIII/Actin, ATG3/Actin, HDAC6/Actin and LC3-II/Actin ratios as described in Figure 2 ( n = 3); ( D ) Control and DMD cell lines were transiently transfected with pEGFP-LC3; 24 h after transfection cells were fixed and analyzed with a fluorescence microscope (scale bar: 50 µm). The histogram shows quantification of the number of autophagic vesicles and indicates a statistically significant increase of cells containing more than 50 vesicles ( n = 3); ( E ) Wild type and DMD cell lines were treated (+) or not (−) with a cocktail of lysosomal protease inhibitors (E64D and <t>Pepstatin</t> A) during 19 h. 10 µg of total protein extracts of each cell lines were separated by SDS-PAGE and analyzed by immunoblot using a specific antibody against LC3. This Western bot is representative of three identical experiments; after quantification, the LC3-II/Actin ratios was set at 1.0 for non-treated conditions in W1 cell line. ** p
    Pepstatin A, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Peninsula Laboratories pepstatin
    The autophagic flux is increased in DMD myoblast cell lines. ( A – C ) 10 µg of total protein extracts from control (W1 W2) and DMD (D1 to D4) cell lines were separated by SDS-PAGE and analyzed by immunoblots probed with antibodies directed against ( A ) proteins involved in the initiation phase of the autophagic process: PI3K class III, BECN1 and BCL2; ( B ) proteins involved in autophagosome nucleation/elongation or transport/fusion: FOXO3a, ATG3, ATG9L1, ATG7, ATG5/12, HDAC6; ( C ) LC3-I and its lipidated form LC3-II. Actin is used as a loading control. The histograms show means ± SD of the normalized PI3KIII/Actin, ATG3/Actin, HDAC6/Actin and LC3-II/Actin ratios as described in Figure 2 ( n = 3); ( D ) Control and DMD cell lines were transiently transfected with pEGFP-LC3; 24 h after transfection cells were fixed and analyzed with a fluorescence microscope (scale bar: 50 µm). The histogram shows quantification of the number of autophagic vesicles and indicates a statistically significant increase of cells containing more than 50 vesicles ( n = 3); ( E ) Wild type and DMD cell lines were treated (+) or not (−) with a cocktail of lysosomal protease inhibitors (E64D and <t>Pepstatin</t> A) during 19 h. 10 µg of total protein extracts of each cell lines were separated by SDS-PAGE and analyzed by immunoblot using a specific antibody against LC3. This Western bot is representative of three identical experiments; after quantification, the LC3-II/Actin ratios was set at 1.0 for non-treated conditions in W1 cell line. ** p
    Pepstatin, supplied by Peninsula Laboratories, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH pepstatin a
    Autophagy induction in MDA-MB-231 cells after treatment with BZ. (A) MDA-MB-231 cells were treated with BZ at various concentrations for 48 h. Cellular proteins were separated by 15% SDS-PAGE for LC3B and 11.25% SDS-PAGE for p62. Immunoblotting was performed using anti-LC3B Ab and anti-p62 mAb. (B) MDA-MB-231 cells were treated with 25 nM of BZ for various lengths of time. Cellular proteins were separated by SDS-PAGE as described above and immunoblotted with anti-LC3B Ab and anti-p62 mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control. (C) MDA-MB-231 cells were cultured with/without BZ (25 nM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μg/ml) and <t>pepstatin</t> A (10 μg/ml) for 48 h. Each number indicates the ratio of LC3B-II/LC3B-I and p62/GAPDH as determined by densitometry.
    Pepstatin A, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioShop pepstatin a
    Autophagy induction in MDA-MB-231 cells after treatment with BZ. (A) MDA-MB-231 cells were treated with BZ at various concentrations for 48 h. Cellular proteins were separated by 15% SDS-PAGE for LC3B and 11.25% SDS-PAGE for p62. Immunoblotting was performed using anti-LC3B Ab and anti-p62 mAb. (B) MDA-MB-231 cells were treated with 25 nM of BZ for various lengths of time. Cellular proteins were separated by SDS-PAGE as described above and immunoblotted with anti-LC3B Ab and anti-p62 mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control. (C) MDA-MB-231 cells were cultured with/without BZ (25 nM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μg/ml) and <t>pepstatin</t> A (10 μg/ml) for 48 h. Each number indicates the ratio of LC3B-II/LC3B-I and p62/GAPDH as determined by densitometry.
    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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    Image Search Results


    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: On the other hand, pepstatin A did not influence the digestion of OVA, suggesting that immune suppression resulting from treatment with pepstatin A is not caused by digestion of OVA in the lysosome.

    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: On the other hand, pepstatin A did not influence the digestion of OVA, suggesting that immune suppression resulting from treatment with pepstatin A is not caused by digestion of OVA in the lysosome.

    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: On the other hand, pepstatin A did not influence the digestion of OVA, suggesting that immune suppression resulting from treatment with pepstatin A is not caused by digestion of OVA in the lysosome.

    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: On the other hand, pepstatin A did not influence the digestion of OVA, suggesting that immune suppression resulting from treatment with pepstatin A is not caused by digestion of OVA in the lysosome.

    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: On the other hand, pepstatin A did not influence the digestion of OVA, suggesting that immune suppression resulting from treatment with pepstatin A is not caused by digestion of OVA in the lysosome.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    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

    PES-induced cell death, BID processing and caspase 8 cleavage are reduced by pepstatin A. ( a ) BC3 and BCBL1 mock-treated or treated with PES (20 μ M for 24 h) in the presence or absence of pepstatin A (20 μ M). Cells were counted by trypan-blue exclusion and the mean±S.D. is reported * P -value=0.02, ** P -value=0.02. ( b ) Western blot was also performed concomitantly with the treatments reported above to analyze the processing of Bid and caspase 8. β -Actin was used as loading control and a representative experiment out of three is shown

    Journal: Cell Death & Disease

    Article Title: HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma

    doi: 10.1038/cddis.2013.263

    Figure Lengend Snippet: PES-induced cell death, BID processing and caspase 8 cleavage are reduced by pepstatin A. ( a ) BC3 and BCBL1 mock-treated or treated with PES (20 μ M for 24 h) in the presence or absence of pepstatin A (20 μ M). Cells were counted by trypan-blue exclusion and the mean±S.D. is reported * P -value=0.02, ** P -value=0.02. ( b ) Western blot was also performed concomitantly with the treatments reported above to analyze the processing of Bid and caspase 8. β -Actin was used as loading control and a representative experiment out of three is shown

    Article Snippet: Cell viability BC3 and BCBL1 were plated in 12-well plates in complete medium at a density of 5 × 105 cells/ml and treated with PES (10–30 μ M; Calbiochem; cat. no. 506155), BORT (10 nM; Millennium Pharmaceutical), pepstatin A (20 μ M; Santa Cruz Biotechnology; cat. no. sc-45036) alone or in combination for the indicated time or with DMSO, as control.

    Techniques: Western Blot

    The effect of cathepsin D inhibition on the degradation and activity of extracellular Cys-C and cysteine cathepsins, respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or pepstatin A ( 4 ), grassystatins D and F ( 1 and 3 ) for 3 days at 37 ºC. A, B) The concentration of Cys-C in the 3-day CM was quantified by human Cys-C ELISA. C, D) The activity of cysteine cathepsins was quantified by incubating the 3-day CM in assay buffer (37 °C, pH 5.5) in the presence of fluorogenic substrate. The asterisks denote significance of P

    Journal: Journal of natural products

    Article Title: Grassystatins D–F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer

    doi: 10.1021/acs.jnatprod.7b00551

    Figure Lengend Snippet: The effect of cathepsin D inhibition on the degradation and activity of extracellular Cys-C and cysteine cathepsins, respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or pepstatin A ( 4 ), grassystatins D and F ( 1 and 3 ) for 3 days at 37 ºC. A, B) The concentration of Cys-C in the 3-day CM was quantified by human Cys-C ELISA. C, D) The activity of cysteine cathepsins was quantified by incubating the 3-day CM in assay buffer (37 °C, pH 5.5) in the presence of fluorogenic substrate. The asterisks denote significance of P

    Article Snippet: To examine the inhibitory activity of grassystatin D and F ( 1 , 3 ) and pepstatin A ( 4 , Enzo Life Sciences) against cellular cathepsins (D and E), kinetic assays were carried out using lysates collected from MDA-MB-231.

    Techniques: Inhibition, Activity Assay, Multiple Displacement Amplification, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    The effect of cathepsin D inhibition on the degradation and activity of extracellular PAI-1 and tPA respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or pepstatin A ( 4 ), grassystatins D and F ( 1 and 3 ) for 3 days at 37 ºC. A, B) The concentration of PAI-1 in the 3-day CM was quantified by PAI-1 human ELISA kit. C, D) The activity of tPA was quantified by tPA human chromogenic activity assay. The asterisks denote significance of P

    Journal: Journal of natural products

    Article Title: Grassystatins D–F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer

    doi: 10.1021/acs.jnatprod.7b00551

    Figure Lengend Snippet: The effect of cathepsin D inhibition on the degradation and activity of extracellular PAI-1 and tPA respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or pepstatin A ( 4 ), grassystatins D and F ( 1 and 3 ) for 3 days at 37 ºC. A, B) The concentration of PAI-1 in the 3-day CM was quantified by PAI-1 human ELISA kit. C, D) The activity of tPA was quantified by tPA human chromogenic activity assay. The asterisks denote significance of P

    Article Snippet: To examine the inhibitory activity of grassystatin D and F ( 1 , 3 ) and pepstatin A ( 4 , Enzo Life Sciences) against cellular cathepsins (D and E), kinetic assays were carried out using lysates collected from MDA-MB-231.

    Techniques: Inhibition, Activity Assay, Multiple Displacement Amplification, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    The cellular inhibitory activities of grassystatins D and F ( 1 and 3 ) and pepstatin A ( 4 ) against cathepsin D/E proteases using cell lysates and intact live cells. A) MDA-MB-231 lysates were treated with grassystatins ( 1 and 3 ) or pepstatin A ( 4 ) in the presence of cathepsin D/E fluorogenic substrate. B) MDA-MB-231 cells were treated with pepstatin A ( 4 ), grassystatin D ( 1 ) or F ( 3 ) for 4 h, cells were lysed, and the protease inhibitory activity was analyzed. C) MDA-MB-231 cells were treated with pepstatin A ( 4 ) or grassystatin F ( 3 ) for 4 h, cells were trypsinized, collected by centrifugation then lysed, and the protease inhibitory activity was analyzed. D) MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 6.6 and incubated with either DMSO, pepstatin A ( 4 ), grassystatins D or F ( 1 or 3 ) for 3 days at 37 ºC. The activity of secreted cathepsins D/E was quantified by incubating the 3-day conditioned media (CM) in assay buffer (37 °C, pH 3.5) in the presence of fluorogenic substrate. The asterisks denote significance of P

    Journal: Journal of natural products

    Article Title: Grassystatins D–F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer

    doi: 10.1021/acs.jnatprod.7b00551

    Figure Lengend Snippet: The cellular inhibitory activities of grassystatins D and F ( 1 and 3 ) and pepstatin A ( 4 ) against cathepsin D/E proteases using cell lysates and intact live cells. A) MDA-MB-231 lysates were treated with grassystatins ( 1 and 3 ) or pepstatin A ( 4 ) in the presence of cathepsin D/E fluorogenic substrate. B) MDA-MB-231 cells were treated with pepstatin A ( 4 ), grassystatin D ( 1 ) or F ( 3 ) for 4 h, cells were lysed, and the protease inhibitory activity was analyzed. C) MDA-MB-231 cells were treated with pepstatin A ( 4 ) or grassystatin F ( 3 ) for 4 h, cells were trypsinized, collected by centrifugation then lysed, and the protease inhibitory activity was analyzed. D) MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 6.6 and incubated with either DMSO, pepstatin A ( 4 ), grassystatins D or F ( 1 or 3 ) for 3 days at 37 ºC. The activity of secreted cathepsins D/E was quantified by incubating the 3-day conditioned media (CM) in assay buffer (37 °C, pH 3.5) in the presence of fluorogenic substrate. The asterisks denote significance of P

    Article Snippet: To examine the inhibitory activity of grassystatin D and F ( 1 , 3 ) and pepstatin A ( 4 , Enzo Life Sciences) against cellular cathepsins (D and E), kinetic assays were carried out using lysates collected from MDA-MB-231.

    Techniques: Multiple Displacement Amplification, Activity Assay, Centrifugation, Incubation

    The effect of cathepsin D inhibitors and siCTSD on the migration of MDA-MB-231 cells. A) MDA-MB-231 cells were incubated for 48 h in the presence of 5 μM cathepsin D inhibitors (grassystatins D and F ( 1 and 3 ) and pepstatin A ( 4 )) and the effect was compared to the solvent control. B) MDA-MB-231 cells were incubated in the presence of 20 nM siCTSD and the effect was compared to the negative control. The graph represents number of migrated cells in each treatment group. The asterisks denote significance of P

    Journal: Journal of natural products

    Article Title: Grassystatins D–F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer

    doi: 10.1021/acs.jnatprod.7b00551

    Figure Lengend Snippet: The effect of cathepsin D inhibitors and siCTSD on the migration of MDA-MB-231 cells. A) MDA-MB-231 cells were incubated for 48 h in the presence of 5 μM cathepsin D inhibitors (grassystatins D and F ( 1 and 3 ) and pepstatin A ( 4 )) and the effect was compared to the solvent control. B) MDA-MB-231 cells were incubated in the presence of 20 nM siCTSD and the effect was compared to the negative control. The graph represents number of migrated cells in each treatment group. The asterisks denote significance of P

    Article Snippet: To examine the inhibitory activity of grassystatin D and F ( 1 , 3 ) and pepstatin A ( 4 , Enzo Life Sciences) against cellular cathepsins (D and E), kinetic assays were carried out using lysates collected from MDA-MB-231.

    Techniques: Migration, Multiple Displacement Amplification, Incubation, Negative Control

    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

    CIT induced autophagy-dependent apoptosis in HepG2 cells. Caspase-3 activity was determined using colorimetric method and was expressed as a percent of control activity. ( A ) HepG2 cells were treated with 5 µM CIT for 6 h, 12 h, or 24 h. ( B ) HepG2 cells were transfected with either 50 nM siRNA against human Atg5 (Si Atg5) or scrambled control siRNA (Si Control), and then treated with 5 µM CIT for 24 h. ( C ) HepG2 cells were pretreated with 40 µM pepstatin A for 4 h, and subsequently treated with 5 µM CIT for 24 h. ** p

    Journal: Toxins

    Article Title: Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells

    doi: 10.3390/toxins7083030

    Figure Lengend Snippet: CIT induced autophagy-dependent apoptosis in HepG2 cells. Caspase-3 activity was determined using colorimetric method and was expressed as a percent of control activity. ( A ) HepG2 cells were treated with 5 µM CIT for 6 h, 12 h, or 24 h. ( B ) HepG2 cells were transfected with either 50 nM siRNA against human Atg5 (Si Atg5) or scrambled control siRNA (Si Control), and then treated with 5 µM CIT for 24 h. ( C ) HepG2 cells were pretreated with 40 µM pepstatin A for 4 h, and subsequently treated with 5 µM CIT for 24 h. ** p

    Article Snippet: HepG2 cells were treated with 5 µM CIT for 6 h, 12 h, or 24 h. Before incubation with 5 µM CIT for 24 h, the cells were pretreated with 50 nM siRNA against Atg5 for 48 h or with 40 µM pepstatin A (Amresco, Solon, OH, USA), a cathepsin D inhibitor, for 4 h, respectively.

    Techniques: Activity Assay, Transfection

    Cathepsin D expression (a) and activity (b) in the jejunum of control (open bars) and methotrexate (MTX)-treated rats receiving vehicle (closed bars), pepstatin A (dark grey bars) or E64 (grey bars). Values are means ± standard error of the mean

    Journal: Clinical and Experimental Immunology

    Article Title: Beneficial effects of cathepsin inhibition to prevent chemotherapy-induced intestinal mucositis

    doi: 10.1111/j.1365-2249.2010.04220.x

    Figure Lengend Snippet: Cathepsin D expression (a) and activity (b) in the jejunum of control (open bars) and methotrexate (MTX)-treated rats receiving vehicle (closed bars), pepstatin A (dark grey bars) or E64 (grey bars). Values are means ± standard error of the mean

    Article Snippet: From D0 to D3, rats also received intraperitoneal injections of pepstatin A (10 mg/kg; Bachem, Weil am Rhein, Germany), E64 (5 mg/kg; Sigma Aldrich, Saint Quentin Fallavier, France) or vehicle, as described previously [ ].

    Techniques: Expressing, Activity Assay

    Chymtrypsin-like (a) and peptidase (b) activities of proteasome in the jejunum of control (open bars) and methotrexate (MTX)-treated rats receiving vehicle (closed bars), pepstatin A (dark grey bars) or E64 (grey bars). Values are means ± standard

    Journal: Clinical and Experimental Immunology

    Article Title: Beneficial effects of cathepsin inhibition to prevent chemotherapy-induced intestinal mucositis

    doi: 10.1111/j.1365-2249.2010.04220.x

    Figure Lengend Snippet: Chymtrypsin-like (a) and peptidase (b) activities of proteasome in the jejunum of control (open bars) and methotrexate (MTX)-treated rats receiving vehicle (closed bars), pepstatin A (dark grey bars) or E64 (grey bars). Values are means ± standard

    Article Snippet: From D0 to D3, rats also received intraperitoneal injections of pepstatin A (10 mg/kg; Bachem, Weil am Rhein, Germany), E64 (5 mg/kg; Sigma Aldrich, Saint Quentin Fallavier, France) or vehicle, as described previously [ ].

    Techniques:

    Food intake (a) and body weight changes (b) in control (closed circles) and methotrexate (MTX)-treated rats receiving vehicle (open circles), pepstatin A (open triangles) or E64 (closed triangles). Values are means ± standard error of the mean

    Journal: Clinical and Experimental Immunology

    Article Title: Beneficial effects of cathepsin inhibition to prevent chemotherapy-induced intestinal mucositis

    doi: 10.1111/j.1365-2249.2010.04220.x

    Figure Lengend Snippet: Food intake (a) and body weight changes (b) in control (closed circles) and methotrexate (MTX)-treated rats receiving vehicle (open circles), pepstatin A (open triangles) or E64 (closed triangles). Values are means ± standard error of the mean

    Article Snippet: From D0 to D3, rats also received intraperitoneal injections of pepstatin A (10 mg/kg; Bachem, Weil am Rhein, Germany), E64 (5 mg/kg; Sigma Aldrich, Saint Quentin Fallavier, France) or vehicle, as described previously [ ].

    Techniques:

    Effects of P140 on autophagy in U-251 MG glioblastoma cells. U-251 MG cells were treated or not with 10 μM P140 for 8 h in the presence or absence of lysosomal enzyme inhibitors pepstatin A (PepA) and E64D. Protein lysates were separated on 4–20% SDS-gels and then transferred onto nitrocellulose membrane. (A) Proteins were revealed with Abs to ATG12/5, MAP1LC3B, SQSTM1, BECLIN-1, and LAMP2A. Comparisons were made between untreated and P140-treated U-251 MG cells. Normalization was performed by measuring total protein directly on the membrane (stain-free procedure). (B) Autophagic markers MAP1LC3B and SQSTM1 measured in the absence and presence of anti-proteases as indicators of flux. The effect of P140 peptide on the flux intensity is shown. The results are displayed with cells collected after 1–12 cycles of divisions or after a longer period of more than 12–20 cycles of division in culture medium. (C) Effect of P140 on ATG12/5, BECLIN-1, and LAMP2A protein expression. Error bars are SEM. Each sample was tested in triplicates or quadruplets in at least 3 independent experiments (1 point corresponds to the mean value of replicates). P values are from Mann-Whitney U tests.

    Journal: Frontiers in Immunology

    Article Title: The Mitochondrion-lysosome Axis in Adaptive and Innate Immunity: Effect of Lupus Regulator Peptide P140 on Mitochondria Autophagy and NETosis

    doi: 10.3389/fimmu.2018.02158

    Figure Lengend Snippet: Effects of P140 on autophagy in U-251 MG glioblastoma cells. U-251 MG cells were treated or not with 10 μM P140 for 8 h in the presence or absence of lysosomal enzyme inhibitors pepstatin A (PepA) and E64D. Protein lysates were separated on 4–20% SDS-gels and then transferred onto nitrocellulose membrane. (A) Proteins were revealed with Abs to ATG12/5, MAP1LC3B, SQSTM1, BECLIN-1, and LAMP2A. Comparisons were made between untreated and P140-treated U-251 MG cells. Normalization was performed by measuring total protein directly on the membrane (stain-free procedure). (B) Autophagic markers MAP1LC3B and SQSTM1 measured in the absence and presence of anti-proteases as indicators of flux. The effect of P140 peptide on the flux intensity is shown. The results are displayed with cells collected after 1–12 cycles of divisions or after a longer period of more than 12–20 cycles of division in culture medium. (C) Effect of P140 on ATG12/5, BECLIN-1, and LAMP2A protein expression. Error bars are SEM. Each sample was tested in triplicates or quadruplets in at least 3 independent experiments (1 point corresponds to the mean value of replicates). P values are from Mann-Whitney U tests.

    Article Snippet: To measure the autophagic flux, half of P140-samples and controls were incubated with 5 μg/mL of each pepstatin A, a potent inhibitor of aspartyl proteases (Merck, 5318) and E-64d/Aloxistatin, a pan-cysteine cathepsin inhibitor (Merck, E8640).

    Techniques: Staining, Expressing, MANN-WHITNEY

    Calorimetric titrations of wild-type HIV-1 protease with Amprenavir ( left panel), acetyl pepstatin ( center panel), and with Amprenavir in the presence of acetyl pepstatin (displacement titration; right panel). These experiments were performed at 25°C in 10 mM acetate, pH 5, 2% DMSO. In all experiments the protease was in the calorimeter reaction cell and the inhibitors in the injection syringe. The inhibitor was added in stepwise injections of 10 μL. For each experiment the reactant concentrations were: ( left panel) 23.7 μM protease, 250 μM Amprenavir; ( center panel) 18.9 μM protease, 300 μM acetyl pepstatin; ( right panel) 19.1 μM protease, 300 μM acetyl pepstatin, 250 μM Amprenavir.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Overcoming drug resistance in HIV-1 chemotherapy: The binding thermodynamics of Amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease

    doi:

    Figure Lengend Snippet: Calorimetric titrations of wild-type HIV-1 protease with Amprenavir ( left panel), acetyl pepstatin ( center panel), and with Amprenavir in the presence of acetyl pepstatin (displacement titration; right panel). These experiments were performed at 25°C in 10 mM acetate, pH 5, 2% DMSO. In all experiments the protease was in the calorimeter reaction cell and the inhibitors in the injection syringe. The inhibitor was added in stepwise injections of 10 μL. For each experiment the reactant concentrations were: ( left panel) 23.7 μM protease, 250 μM Amprenavir; ( center panel) 18.9 μM protease, 300 μM acetyl pepstatin; ( right panel) 19.1 μM protease, 300 μM acetyl pepstatin, 250 μM Amprenavir.

    Article Snippet: The enzyme solution in the calorimetric cell was titrated with Amprenavir, TMC-126, or acetyl pepstatin (Bachem AG) dissolved in the same buffer.

    Techniques: Titration, Injection

    Calorimetric titrations of wild-type HIV-1 protease with the inhibitor TMC-126 ( left panel), the inhibitor acetyl pepstatin ( center ), and with TMC-126 in the presence of acetyl pepstatin (displacement titration; right panel). These experiments were performed at 25°C in 10 mM acetate, pH 5, 2% DMSO. In all experiments the protease was in the calorimeter reaction cell and the inhibitors in the injection syringe. The inhibitor was added in stepwise injections of 10 μL. For each experiment the reactant concentrations were: ( left panel) 6.4 μM protease, 84 μM TMC-126; ( center panel) 18.9 μM protease, 300 μM acetyl pepstatin; ( right panel) 6.6 μM protease, 400 μM acetyl pepstatin, 53 μM TMC-126.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Overcoming drug resistance in HIV-1 chemotherapy: The binding thermodynamics of Amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease

    doi:

    Figure Lengend Snippet: Calorimetric titrations of wild-type HIV-1 protease with the inhibitor TMC-126 ( left panel), the inhibitor acetyl pepstatin ( center ), and with TMC-126 in the presence of acetyl pepstatin (displacement titration; right panel). These experiments were performed at 25°C in 10 mM acetate, pH 5, 2% DMSO. In all experiments the protease was in the calorimeter reaction cell and the inhibitors in the injection syringe. The inhibitor was added in stepwise injections of 10 μL. For each experiment the reactant concentrations were: ( left panel) 6.4 μM protease, 84 μM TMC-126; ( center panel) 18.9 μM protease, 300 μM acetyl pepstatin; ( right panel) 6.6 μM protease, 400 μM acetyl pepstatin, 53 μM TMC-126.

    Article Snippet: The enzyme solution in the calorimetric cell was titrated with Amprenavir, TMC-126, or acetyl pepstatin (Bachem AG) dissolved in the same buffer.

    Techniques: Titration, Injection

    hnf4 is required for blood feeding a , Graph of the aspartyl protease activity of lysates from control(RNAi) or hnf4(RNAi) parasites as determined by the ability to cleave the fluorogenic substrate, mca-GKPILFFRLK-K(dnp) in the presence of no inhibitor (DMSO), the general cysteine protease inhibitor E-64 (E-64), or the aspartyl protease inhibitor pepstatin A (pepstatin). b , Graph quantifying the recovery rate of worms from transplant recipients. Data are from five recipients. c , Representative photographs of livers of mice 30 days after transplant with RNAi-treated parasites. The number of livers grossly similar to the representative photograph is indicated in the upper right each panel. Data are from two recipients in one biological replicate. d , Graph showing quantification of worm length from Fig. 4f . n = 15 for control(RNAi) male parasites and 16 hnf4(RNAi) male parasites from 3 separate recipients. Scale bar, c , 1cm. ns, not significant, ****, p

    Journal: bioRxiv

    Article Title: A single-cell RNAseq atlas of the pathogenic stage of Schistosoma mansoni identifies a key regulator of blood feeding

    doi: 10.1101/2020.02.03.932004

    Figure Lengend Snippet: hnf4 is required for blood feeding a , Graph of the aspartyl protease activity of lysates from control(RNAi) or hnf4(RNAi) parasites as determined by the ability to cleave the fluorogenic substrate, mca-GKPILFFRLK-K(dnp) in the presence of no inhibitor (DMSO), the general cysteine protease inhibitor E-64 (E-64), or the aspartyl protease inhibitor pepstatin A (pepstatin). b , Graph quantifying the recovery rate of worms from transplant recipients. Data are from five recipients. c , Representative photographs of livers of mice 30 days after transplant with RNAi-treated parasites. The number of livers grossly similar to the representative photograph is indicated in the upper right each panel. Data are from two recipients in one biological replicate. d , Graph showing quantification of worm length from Fig. 4f . n = 15 for control(RNAi) male parasites and 16 hnf4(RNAi) male parasites from 3 separate recipients. Scale bar, c , 1cm. ns, not significant, ****, p

    Article Snippet: Pepstatin A (MP Biomedicals, 0219536805) and E-64 controls were set up by incubating the sample with 10µM of either inhibitor for 30 minutes at room temperature.

    Techniques: Activity Assay, Protease Inhibitor, Mouse Assay

    The autophagic flux is increased in DMD myoblast cell lines. ( A – C ) 10 µg of total protein extracts from control (W1 W2) and DMD (D1 to D4) cell lines were separated by SDS-PAGE and analyzed by immunoblots probed with antibodies directed against ( A ) proteins involved in the initiation phase of the autophagic process: PI3K class III, BECN1 and BCL2; ( B ) proteins involved in autophagosome nucleation/elongation or transport/fusion: FOXO3a, ATG3, ATG9L1, ATG7, ATG5/12, HDAC6; ( C ) LC3-I and its lipidated form LC3-II. Actin is used as a loading control. The histograms show means ± SD of the normalized PI3KIII/Actin, ATG3/Actin, HDAC6/Actin and LC3-II/Actin ratios as described in Figure 2 ( n = 3); ( D ) Control and DMD cell lines were transiently transfected with pEGFP-LC3; 24 h after transfection cells were fixed and analyzed with a fluorescence microscope (scale bar: 50 µm). The histogram shows quantification of the number of autophagic vesicles and indicates a statistically significant increase of cells containing more than 50 vesicles ( n = 3); ( E ) Wild type and DMD cell lines were treated (+) or not (−) with a cocktail of lysosomal protease inhibitors (E64D and Pepstatin A) during 19 h. 10 µg of total protein extracts of each cell lines were separated by SDS-PAGE and analyzed by immunoblot using a specific antibody against LC3. This Western bot is representative of three identical experiments; after quantification, the LC3-II/Actin ratios was set at 1.0 for non-treated conditions in W1 cell line. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Modulation of Protein Quality Control and Proteasome to Autophagy Switch in Immortalized Myoblasts from Duchenne Muscular Dystrophy Patients

    doi: 10.3390/ijms19010178

    Figure Lengend Snippet: The autophagic flux is increased in DMD myoblast cell lines. ( A – C ) 10 µg of total protein extracts from control (W1 W2) and DMD (D1 to D4) cell lines were separated by SDS-PAGE and analyzed by immunoblots probed with antibodies directed against ( A ) proteins involved in the initiation phase of the autophagic process: PI3K class III, BECN1 and BCL2; ( B ) proteins involved in autophagosome nucleation/elongation or transport/fusion: FOXO3a, ATG3, ATG9L1, ATG7, ATG5/12, HDAC6; ( C ) LC3-I and its lipidated form LC3-II. Actin is used as a loading control. The histograms show means ± SD of the normalized PI3KIII/Actin, ATG3/Actin, HDAC6/Actin and LC3-II/Actin ratios as described in Figure 2 ( n = 3); ( D ) Control and DMD cell lines were transiently transfected with pEGFP-LC3; 24 h after transfection cells were fixed and analyzed with a fluorescence microscope (scale bar: 50 µm). The histogram shows quantification of the number of autophagic vesicles and indicates a statistically significant increase of cells containing more than 50 vesicles ( n = 3); ( E ) Wild type and DMD cell lines were treated (+) or not (−) with a cocktail of lysosomal protease inhibitors (E64D and Pepstatin A) during 19 h. 10 µg of total protein extracts of each cell lines were separated by SDS-PAGE and analyzed by immunoblot using a specific antibody against LC3. This Western bot is representative of three identical experiments; after quantification, the LC3-II/Actin ratios was set at 1.0 for non-treated conditions in W1 cell line. ** p

    Article Snippet: Pepstatin A was from Merck-Millipore (Burlington, MA, USA).

    Techniques: SDS Page, Western Blot, Transfection, Fluorescence, Microscopy

    Autophagy induction in MDA-MB-231 cells after treatment with BZ. (A) MDA-MB-231 cells were treated with BZ at various concentrations for 48 h. Cellular proteins were separated by 15% SDS-PAGE for LC3B and 11.25% SDS-PAGE for p62. Immunoblotting was performed using anti-LC3B Ab and anti-p62 mAb. (B) MDA-MB-231 cells were treated with 25 nM of BZ for various lengths of time. Cellular proteins were separated by SDS-PAGE as described above and immunoblotted with anti-LC3B Ab and anti-p62 mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control. (C) MDA-MB-231 cells were cultured with/without BZ (25 nM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μg/ml) and pepstatin A (10 μg/ml) for 48 h. Each number indicates the ratio of LC3B-II/LC3B-I and p62/GAPDH as determined by densitometry.

    Journal: International Journal of Oncology

    Article Title: Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells

    doi: 10.3892/ijo.2011.1317

    Figure Lengend Snippet: Autophagy induction in MDA-MB-231 cells after treatment with BZ. (A) MDA-MB-231 cells were treated with BZ at various concentrations for 48 h. Cellular proteins were separated by 15% SDS-PAGE for LC3B and 11.25% SDS-PAGE for p62. Immunoblotting was performed using anti-LC3B Ab and anti-p62 mAb. (B) MDA-MB-231 cells were treated with 25 nM of BZ for various lengths of time. Cellular proteins were separated by SDS-PAGE as described above and immunoblotted with anti-LC3B Ab and anti-p62 mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control. (C) MDA-MB-231 cells were cultured with/without BZ (25 nM) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μg/ml) and pepstatin A (10 μg/ml) for 48 h. Each number indicates the ratio of LC3B-II/LC3B-I and p62/GAPDH as determined by densitometry.

    Article Snippet: E-64d and Pepstatin A, which are inhibitors of lysosomal proteases, were purchased from Biomol International LP (Plymouth Meeting, PA, USA).

    Techniques: Multiple Displacement Amplification, SDS Page, Cell Culture

    Immunoblotting with anti-LC3B Ab and anti-p62 Ab after treatment with CAM in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with CAM (50 μg/ml) for various lengths of time. (B) MDA-MB-231 cells were cultured with CAM (25 and 50 μg/ml) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μg/ml) and pepstatin A (10 μg/ml) for 48 h. Cellular proteins were separated by 15% SDS-PAGE for LC3B and 11.25% SDS-PAGE for p62, and immunoblotted with anti-LC3B Ab and anti-p62 mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control. Each number indicates the ratio of LC3B-II/LC3B-I and p62/GAPDH as determined by densitometry.

    Journal: International Journal of Oncology

    Article Title: Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells

    doi: 10.3892/ijo.2011.1317

    Figure Lengend Snippet: Immunoblotting with anti-LC3B Ab and anti-p62 Ab after treatment with CAM in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with CAM (50 μg/ml) for various lengths of time. (B) MDA-MB-231 cells were cultured with CAM (25 and 50 μg/ml) in the presence or absence of lysosomal inhibitors (LI), E-64d (10 μg/ml) and pepstatin A (10 μg/ml) for 48 h. Cellular proteins were separated by 15% SDS-PAGE for LC3B and 11.25% SDS-PAGE for p62, and immunoblotted with anti-LC3B Ab and anti-p62 mAb. Immunoblotting with anti-GAPDH mAb was performed as an internal control. Each number indicates the ratio of LC3B-II/LC3B-I and p62/GAPDH as determined by densitometry.

    Article Snippet: E-64d and Pepstatin A, which are inhibitors of lysosomal proteases, were purchased from Biomol International LP (Plymouth Meeting, PA, USA).

    Techniques: Chick Chorioallantoic Membrane Assay, Multiple Displacement Amplification, Cell Culture, SDS Page