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  • 94
    Thermo Fisher pepstatin a
    SiNPs enlarge lysosomes and inhibit lysosomal acidification. a  A549 cells transiently expressing YFP-LAMP1 were treated with SiNPs at 50 μg/mL for 24 h. The cells were then fixed and imaged by confocal microscopy. The 3D structure of the lysosome were restructured with Imaris software. Scale bars: 4 μm.  b  The lysosome volumes of the cells treated as in ( a ) were quantified, and the difference between control and SiNPs treatment groups was tested with a Wilcoxon rank sum test.  c  A549 cells were treated with SiNPs at 50 μg/mL for 24 h, then stained with Lysosensor Green DND-189. The lysosensor fluorescence intensity was detected by flow cytometry. A representative flow cytometry profile is shown on the left. Quantification shown on the right represents the fluorescence intensity of the lysosensor probe.  d  A549 cells treated with SiNPs at 50 μg/mL for 24 h were incubated with 1.0 mM Bodipy-FL-pepstatin A for 1 h, and then visualized by confocal microscopy. Scale bars: 5 μm.  e  The mature CTSD protein level was assessed by western blot after SiNPs treatment as in ( d ). The bottom panel shows the statistics for relative mature-CTSD protein level. Data are presented as mean ± SEM, ** P
    Pepstatin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pepstatin a
    The use of protease inhibitors implicates plasmin in the activation of pro-MMP-2 in glioma–astrocyte cocultures. A , The addition of TIMP-1 and -2 did not affect the activation of MMP-2 in glioma–astrocyte cocultures, thereby excluding the participation of MMPs. Amixture of TIMP-1 (2 μg/ml) and TIMP-2 (2 μg/ml) was also ineffective at preventing pro-MMP-2 activation (data not shown). B , Shown are representative gelatin zymograms of conditioned media collected after 24 hr for U251N–astrocyte cocultures with (20 μg/ml) or without (Control) protease inhibitors; each condition was assessed in duplicate. <t>Pepstatin</t> A and E-64 did not block pro-MMP-2 activation, whereas aprotinin, an inhibitor of serine proteinases, and α 2 -antiplasmin (an inhibitor of the serine proteinase plasmin) attenuated active MMP-2 levels. These results were replicated in five experiments. C , The level of active MMP-2 generated in five cocultures of U251N–astrocyte was normalized to control cultures; then the level of active MMP-2 in treated cultures was expressed as a percentage of this normalized control. Values are the mean ± SEM; *** p
    Pepstatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 23406 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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    Santa Cruz Biotechnology pepstatin a
    4E-BP1 mediates rapamycin-induced autophagy that inhibits rotavirus infection. (A) Caco2 cells transduced with lentiviral particles carrying a construct of TagGFP2-LC3 driven by the elongation factor-1 promotor were cultured at 37°C for 48 h in DMEM medium containing EBSS medium containing 1mM <t>pepstatin</t> A and E-64-d solution (for starvation) and 10 nM rapamycin. LC3-positive puncta was observed by confocal laser microscopy. (B) LC3-I and LC3-II protein levels were examined by western blot analysis. Protein samples were extracted from Caco2 cells treated with indicated concentrations of rapamycin (48 h). Quantification of the intensity of the immunoreactive bands of both LC3-I and LC3-II was performed using Odyssey V3.0 software. Densitometric analysis of immunoblots of LC3 was expressed as the ratio of LC3-II to LC3-I, and the ratio of LC3II/LC3I was expressed in arbitrary units. (C) Western blot visualized LC3-I and LC3-II protein levels in starvation (EBSS medium containing 1mM pepstatin A and E-64-d solution) treated Caco2 cells. The ratio of LC3II/LC3I was expressed in arbitrary units. (D) Starvation significantly inhibited rotavirus RNA in Caco2 cells (n = 6, mean ± SEM, *P
    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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    Millipore pepstatin a p5318
    4E-BP1 mediates rapamycin-induced autophagy that inhibits rotavirus infection. (A) Caco2 cells transduced with lentiviral particles carrying a construct of TagGFP2-LC3 driven by the elongation factor-1 promotor were cultured at 37°C for 48 h in DMEM medium containing EBSS medium containing 1mM <t>pepstatin</t> A and E-64-d solution (for starvation) and 10 nM rapamycin. LC3-positive puncta was observed by confocal laser microscopy. (B) LC3-I and LC3-II protein levels were examined by western blot analysis. Protein samples were extracted from Caco2 cells treated with indicated concentrations of rapamycin (48 h). Quantification of the intensity of the immunoreactive bands of both LC3-I and LC3-II was performed using Odyssey V3.0 software. Densitometric analysis of immunoblots of LC3 was expressed as the ratio of LC3-II to LC3-I, and the ratio of LC3II/LC3I was expressed in arbitrary units. (C) Western blot visualized LC3-I and LC3-II protein levels in starvation (EBSS medium containing 1mM pepstatin A and E-64-d solution) treated Caco2 cells. The ratio of LC3II/LC3I was expressed in arbitrary units. (D) Starvation significantly inhibited rotavirus RNA in Caco2 cells (n = 6, mean ± SEM, *P
    Pepstatin A P5318, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem pepstatin a
    Inhibition of autophagy rescues cell death in Mar-treated PC3 cells. The cell death of PC3 cells exposed to Mar alone or combined treatment. Left panel, cell death by PI exclusion. Right panel, immunoblot analysis of LC3BII, Atg5 and p62 levels in PC3 cells after treatments. OD of LC3BII relative to the control was quantified as mean±S.D., n =3. ( a ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 5 mM 3-MA for 24 h. ( b ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 10 μ M E-64D/10 μ M <t>Pepstatin</t> A (Pep) for 24 h. ( c ) Knockdown of Atg5 by siRNA for 24 or 48 h in PC3 cells and then treated with 10 μ M Mar. ( d ) Knockdown of LC3B by siRNA for 24 or 48 h in PC3 cells prior to Mar treatment. ** 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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    Applichem pepstatin a
    Inhibition of autophagy rescues cell death in Mar-treated PC3 cells. The cell death of PC3 cells exposed to Mar alone or combined treatment. Left panel, cell death by PI exclusion. Right panel, immunoblot analysis of LC3BII, Atg5 and p62 levels in PC3 cells after treatments. OD of LC3BII relative to the control was quantified as mean±S.D., n =3. ( a ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 5 mM 3-MA for 24 h. ( b ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 10 μ M E-64D/10 μ M <t>Pepstatin</t> A (Pep) for 24 h. ( c ) Knockdown of Atg5 by siRNA for 24 or 48 h in PC3 cells and then treated with 10 μ M Mar. ( d ) Knockdown of LC3B by siRNA for 24 or 48 h in PC3 cells prior to Mar treatment. ** 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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    Bio-Rad pepstatin
    Inhibition of autophagy rescues cell death in Mar-treated PC3 cells. The cell death of PC3 cells exposed to Mar alone or combined treatment. Left panel, cell death by PI exclusion. Right panel, immunoblot analysis of LC3BII, Atg5 and p62 levels in PC3 cells after treatments. OD of LC3BII relative to the control was quantified as mean±S.D., n =3. ( a ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 5 mM 3-MA for 24 h. ( b ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 10 μ M E-64D/10 μ M <t>Pepstatin</t> A (Pep) for 24 h. ( c ) Knockdown of Atg5 by siRNA for 24 or 48 h in PC3 cells and then treated with 10 μ M Mar. ( d ) Knockdown of LC3B by siRNA for 24 or 48 h in PC3 cells prior to Mar treatment. ** P
    Pepstatin, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    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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    Thermo Fisher bodipy fl pepstatin a
    P2X7R activation reduced cathepsin D binding and increased lipid oxidation. A ) Stimulation with BzATP (200 μM) inhibited binding of <t>Bodipy</t> FL <t>pepstatin</t> A to active cathepsin D in ARPE-19 cells. * P
    Bodipy Fl Pepstatin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    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
    EWS-Fli-1 turns over by a lysosome-dependent mechanism: (A) Knockdown of CIMPR or VPS26A results in destabilization of FLAG-EWS-Fli-1. 293 cells were cotransfected with FLAG-EWS-Fli-1 and shRNA against luciferase (control), CIMPR, or VPS26A. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (B) TFEB induces EWS-Fli-1 degradation in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and HA-TFEB or empty vector. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (C) Chloroquine stabilizes EWS-Fli-1 in 293 cells. 293 cells were transfected with FLAG-EWS-Fli-1. Transfected cells were left untreated (control) or treated with 100 μM chloroquine for 12 h. The levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (D) Cathepsin D degrades EWS-Fli-1, but not p53, in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and cathepsin D or empty vector. Transfected cells were left untreated or treated with 100 μM chloroquine for 12 h or 100 nM <t>pepstatin</t> A for 12 h. 293 cells were cotransfected with FLAG-p53 and cathepsin D or empty vector. The levels of FLAG-EWS-Fli-1 and FLAG-p53 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (E) Cathepsin D degrades endogenous EWS-Fli-1 in A673 Ewing sarcoma cells. A673 cells were infected with a lentivirus vector expressing cathepsin D or an empty vector, the infected cells were selected with puromycin, and the levels of endogenous EWS-Fli-1 were examined by anti-Fli-1 C-terminus antibody immunoblotting at 4 days after infection. Nucleolin serves as a loading control. (F) Chloroquine stabilizes endogenous EWS-Fli-1 in A673 cells. A673 cells were left untreated, treated with 100 μM chloroquine for 12 h, or treated with 10 μM MG-132 for 12 h. The levels of EWS-Fli-1 were examined by anti-Fli-1 C-terminus immunoblotting. While chloroquine increased the levels of endogenous EWS-Fli-1, MG-132 had no effect on the EWS-Fli-1 protein levels, suggesting that EWS-Fli-1 turns over by a lysosomal, but not proteasomal mechanism. (G) Endogenous EWS-Fli-1 in A673 cells displays increased lysosomal location upon chloroquine treatment. A673 cells were treated with 100 μM chloroquine for 12 h or left untreated, and the whole cell extract (WCE) and lysosomal fraction were isolated. The abundance of EWS-Fli-1, LAMP2, p62/SQSTM1, and mSin3A in each fraction was determined by immunoblotting.
    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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    Peninsula Laboratories pepstatin a
    EWS-Fli-1 turns over by a lysosome-dependent mechanism: (A) Knockdown of CIMPR or VPS26A results in destabilization of FLAG-EWS-Fli-1. 293 cells were cotransfected with FLAG-EWS-Fli-1 and shRNA against luciferase (control), CIMPR, or VPS26A. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (B) TFEB induces EWS-Fli-1 degradation in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and HA-TFEB or empty vector. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (C) Chloroquine stabilizes EWS-Fli-1 in 293 cells. 293 cells were transfected with FLAG-EWS-Fli-1. Transfected cells were left untreated (control) or treated with 100 μM chloroquine for 12 h. The levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (D) Cathepsin D degrades EWS-Fli-1, but not p53, in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and cathepsin D or empty vector. Transfected cells were left untreated or treated with 100 μM chloroquine for 12 h or 100 nM <t>pepstatin</t> A for 12 h. 293 cells were cotransfected with FLAG-p53 and cathepsin D or empty vector. The levels of FLAG-EWS-Fli-1 and FLAG-p53 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (E) Cathepsin D degrades endogenous EWS-Fli-1 in A673 Ewing sarcoma cells. A673 cells were infected with a lentivirus vector expressing cathepsin D or an empty vector, the infected cells were selected with puromycin, and the levels of endogenous EWS-Fli-1 were examined by anti-Fli-1 C-terminus antibody immunoblotting at 4 days after infection. Nucleolin serves as a loading control. (F) Chloroquine stabilizes endogenous EWS-Fli-1 in A673 cells. A673 cells were left untreated, treated with 100 μM chloroquine for 12 h, or treated with 10 μM MG-132 for 12 h. The levels of EWS-Fli-1 were examined by anti-Fli-1 C-terminus immunoblotting. While chloroquine increased the levels of endogenous EWS-Fli-1, MG-132 had no effect on the EWS-Fli-1 protein levels, suggesting that EWS-Fli-1 turns over by a lysosomal, but not proteasomal mechanism. (G) Endogenous EWS-Fli-1 in A673 cells displays increased lysosomal location upon chloroquine treatment. A673 cells were treated with 100 μM chloroquine for 12 h or left untreated, and the whole cell extract (WCE) and lysosomal fraction were isolated. The abundance of EWS-Fli-1, LAMP2, p62/SQSTM1, and mSin3A in each fraction was determined by immunoblotting.
    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
    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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    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
    https://www.bioz.com/result/pepstatin a/product/Biomol GmbH
    Average 92 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    pepstatin a - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/pepstatin a/product/BioShop
    Average 92 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    pepstatin a - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    SiNPs enlarge lysosomes and inhibit lysosomal acidification. a  A549 cells transiently expressing YFP-LAMP1 were treated with SiNPs at 50 μg/mL for 24 h. The cells were then fixed and imaged by confocal microscopy. The 3D structure of the lysosome were restructured with Imaris software. Scale bars: 4 μm.  b  The lysosome volumes of the cells treated as in ( a ) were quantified, and the difference between control and SiNPs treatment groups was tested with a Wilcoxon rank sum test.  c  A549 cells were treated with SiNPs at 50 μg/mL for 24 h, then stained with Lysosensor Green DND-189. The lysosensor fluorescence intensity was detected by flow cytometry. A representative flow cytometry profile is shown on the left. Quantification shown on the right represents the fluorescence intensity of the lysosensor probe.  d  A549 cells treated with SiNPs at 50 μg/mL for 24 h were incubated with 1.0 mM Bodipy-FL-pepstatin A for 1 h, and then visualized by confocal microscopy. Scale bars: 5 μm.  e  The mature CTSD protein level was assessed by western blot after SiNPs treatment as in ( d ). The bottom panel shows the statistics for relative mature-CTSD protein level. Data are presented as mean ± SEM, ** P

    Journal: Cell Death & Disease

    Article Title: Autophagic flux blockage in alveolar epithelial cells is essential in silica nanoparticle-induced pulmonary fibrosis

    doi: 10.1038/s41419-019-1340-8

    Figure Lengend Snippet: SiNPs enlarge lysosomes and inhibit lysosomal acidification. a A549 cells transiently expressing YFP-LAMP1 were treated with SiNPs at 50 μg/mL for 24 h. The cells were then fixed and imaged by confocal microscopy. The 3D structure of the lysosome were restructured with Imaris software. Scale bars: 4 μm. b The lysosome volumes of the cells treated as in ( a ) were quantified, and the difference between control and SiNPs treatment groups was tested with a Wilcoxon rank sum test. c A549 cells were treated with SiNPs at 50 μg/mL for 24 h, then stained with Lysosensor Green DND-189. The lysosensor fluorescence intensity was detected by flow cytometry. A representative flow cytometry profile is shown on the left. Quantification shown on the right represents the fluorescence intensity of the lysosensor probe. d A549 cells treated with SiNPs at 50 μg/mL for 24 h were incubated with 1.0 mM Bodipy-FL-pepstatin A for 1 h, and then visualized by confocal microscopy. Scale bars: 5 μm. e The mature CTSD protein level was assessed by western blot after SiNPs treatment as in ( d ). The bottom panel shows the statistics for relative mature-CTSD protein level. Data are presented as mean ± SEM, ** P

    Article Snippet: Bodipy-FL-pepstatin A (P12271) was from Invitrogen.

    Techniques: Expressing, Confocal Microscopy, Software, Staining, Fluorescence, Flow Cytometry, Cytometry, Incubation, Western Blot

    The use of protease inhibitors implicates plasmin in the activation of pro-MMP-2 in glioma–astrocyte cocultures. A , The addition of TIMP-1 and -2 did not affect the activation of MMP-2 in glioma–astrocyte cocultures, thereby excluding the participation of MMPs. Amixture of TIMP-1 (2 μg/ml) and TIMP-2 (2 μg/ml) was also ineffective at preventing pro-MMP-2 activation (data not shown). B , Shown are representative gelatin zymograms of conditioned media collected after 24 hr for U251N–astrocyte cocultures with (20 μg/ml) or without (Control) protease inhibitors; each condition was assessed in duplicate. Pepstatin A and E-64 did not block pro-MMP-2 activation, whereas aprotinin, an inhibitor of serine proteinases, and α 2 -antiplasmin (an inhibitor of the serine proteinase plasmin) attenuated active MMP-2 levels. These results were replicated in five experiments. C , The level of active MMP-2 generated in five cocultures of U251N–astrocyte was normalized to control cultures; then the level of active MMP-2 in treated cultures was expressed as a percentage of this normalized control. Values are the mean ± SEM; *** p

    Journal: The Journal of Neuroscience

    Article Title: Exploitation of Astrocytes by Glioma Cells to Facilitate Invasiveness: A Mechanism Involving Matrix Metalloproteinase-2 and the Urokinase-Type Plasminogen Activator–Plasmin Cascade

    doi: 10.1523/JNEUROSCI.23-10-04034.2003

    Figure Lengend Snippet: The use of protease inhibitors implicates plasmin in the activation of pro-MMP-2 in glioma–astrocyte cocultures. A , The addition of TIMP-1 and -2 did not affect the activation of MMP-2 in glioma–astrocyte cocultures, thereby excluding the participation of MMPs. Amixture of TIMP-1 (2 μg/ml) and TIMP-2 (2 μg/ml) was also ineffective at preventing pro-MMP-2 activation (data not shown). B , Shown are representative gelatin zymograms of conditioned media collected after 24 hr for U251N–astrocyte cocultures with (20 μg/ml) or without (Control) protease inhibitors; each condition was assessed in duplicate. Pepstatin A and E-64 did not block pro-MMP-2 activation, whereas aprotinin, an inhibitor of serine proteinases, and α 2 -antiplasmin (an inhibitor of the serine proteinase plasmin) attenuated active MMP-2 levels. These results were replicated in five experiments. C , The level of active MMP-2 generated in five cocultures of U251N–astrocyte was normalized to control cultures; then the level of active MMP-2 in treated cultures was expressed as a percentage of this normalized control. Values are the mean ± SEM; *** p

    Article Snippet: The following inhibitors were used: pepstatin A, E-64 [ trans -epoxysuccinyl- l -leucylamido-(4-guanidino)-butane], aprotinin (from Sigma-Aldrich, Oakville, CA), α2 -antiplasmin (Calbiochem, La Jolla, CA), and recombinant human tissue inhibitors of metalloproteinases (TIMPs) -1 or -2 (generously provided by Dr. Dylan Edwards, University of East Anglia, Norwich, UK).

    Techniques: Activation Assay, Blocking Assay, Generated

    Growth curves recorded from two cultures grown under different conditions (blue, dashed curve represents standard growing conditions; red, solid curve with addition of protease inhibitor pepstatin). A small time lag between both curves is evident and makes a comparison based on absolute time points inaccurate.

    Journal: International Journal of Proteomics

    Article Title: Miniaturized Mass-Spectrometry-Based Analysis System for Fully Automated Examination of Conditioned Cell Culture Media

    doi: 10.1155/2012/290457

    Figure Lengend Snippet: Growth curves recorded from two cultures grown under different conditions (blue, dashed curve represents standard growing conditions; red, solid curve with addition of protease inhibitor pepstatin). A small time lag between both curves is evident and makes a comparison based on absolute time points inaccurate.

    Article Snippet: In the protease inhibition experiments, 10 μ M pepstatin (Sigma Aldrich) was added to the culture flask after precultivation.

    Techniques: Protease Inhibitor

    Ratio of 881.45 Da fragment to intact mating factor (1682.84 Da plus 1698.83 Da; right scale). No mating factor or fragments are detected in first 4 investigated time points. At later time points ratios between 0.8 and 2 for culture grown under standard conditions were obtained (right scale, green bars). Note significant reduction of relative amount of 881.45 Da fragments in pepstatin-containing culture (blue bars). Dashed, red curve (left, logarithmic scale) plots growth curve of cells.

    Journal: International Journal of Proteomics

    Article Title: Miniaturized Mass-Spectrometry-Based Analysis System for Fully Automated Examination of Conditioned Cell Culture Media

    doi: 10.1155/2012/290457

    Figure Lengend Snippet: Ratio of 881.45 Da fragment to intact mating factor (1682.84 Da plus 1698.83 Da; right scale). No mating factor or fragments are detected in first 4 investigated time points. At later time points ratios between 0.8 and 2 for culture grown under standard conditions were obtained (right scale, green bars). Note significant reduction of relative amount of 881.45 Da fragments in pepstatin-containing culture (blue bars). Dashed, red curve (left, logarithmic scale) plots growth curve of cells.

    Article Snippet: In the protease inhibition experiments, 10 μ M pepstatin (Sigma Aldrich) was added to the culture flask after precultivation.

    Techniques:

    After PC-supported adjustment of measurement series of pepstatin-containing culture a perfect matching of both growth curves was obtained. All points lie on fitted sigmoid function (solid, green curve), allowing a valid comparison.

    Journal: International Journal of Proteomics

    Article Title: Miniaturized Mass-Spectrometry-Based Analysis System for Fully Automated Examination of Conditioned Cell Culture Media

    doi: 10.1155/2012/290457

    Figure Lengend Snippet: After PC-supported adjustment of measurement series of pepstatin-containing culture a perfect matching of both growth curves was obtained. All points lie on fitted sigmoid function (solid, green curve), allowing a valid comparison.

    Article Snippet: In the protease inhibition experiments, 10 μ M pepstatin (Sigma Aldrich) was added to the culture flask after precultivation.

    Techniques:

    ApoL2 does not regulate autophagy. HeLa cells were transfected with siRNA Control 1 (labeled as siC) or siRNA against ApoL2-II (labeled as siApo) for 48 h and then the medium was changed or they were incubated with EBSS for 6 h to induce autophagy. The protease inhibitors pepstatin and E64D (10 μ M each) were used to block autophagic flux. A representative western blot is shown in a . ApoL2 levels were quantified and are shown in b : data were weighted to ponceau or actin and then normalized against Control 1-transfected untreated cells. ( c ) Quantification of relative LC3-II levels: data were weighted to ponceau or actin and then normalized against Control 1-transfected HeLa cells with protease inhibitors as control of basal autophagy. ( d ) Quantification of relative p62 levels: data were weighted to ponceau or actin and then normalized against Control 1-transfected untreated cells. Graphs show average and S.D. of three independent experiments

    Journal: Cell Death & Disease

    Article Title: Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein

    doi: 10.1038/cddis.2014.237

    Figure Lengend Snippet: ApoL2 does not regulate autophagy. HeLa cells were transfected with siRNA Control 1 (labeled as siC) or siRNA against ApoL2-II (labeled as siApo) for 48 h and then the medium was changed or they were incubated with EBSS for 6 h to induce autophagy. The protease inhibitors pepstatin and E64D (10 μ M each) were used to block autophagic flux. A representative western blot is shown in a . ApoL2 levels were quantified and are shown in b : data were weighted to ponceau or actin and then normalized against Control 1-transfected untreated cells. ( c ) Quantification of relative LC3-II levels: data were weighted to ponceau or actin and then normalized against Control 1-transfected HeLa cells with protease inhibitors as control of basal autophagy. ( d ) Quantification of relative p62 levels: data were weighted to ponceau or actin and then normalized against Control 1-transfected untreated cells. Graphs show average and S.D. of three independent experiments

    Article Snippet: Autophagy flux was blocked by adding the protease inhibitors pepstatin A and E64d (Sigma, St. Louis, MO, USA, 10 μ M each) simultaneously with the treatments.

    Techniques: Transfection, Labeling, Incubation, Blocking Assay, Western Blot

    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

    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

    4E-BP1 mediates rapamycin-induced autophagy that inhibits rotavirus infection. (A) Caco2 cells transduced with lentiviral particles carrying a construct of TagGFP2-LC3 driven by the elongation factor-1 promotor were cultured at 37°C for 48 h in DMEM medium containing EBSS medium containing 1mM pepstatin A and E-64-d solution (for starvation) and 10 nM rapamycin. LC3-positive puncta was observed by confocal laser microscopy. (B) LC3-I and LC3-II protein levels were examined by western blot analysis. Protein samples were extracted from Caco2 cells treated with indicated concentrations of rapamycin (48 h). Quantification of the intensity of the immunoreactive bands of both LC3-I and LC3-II was performed using Odyssey V3.0 software. Densitometric analysis of immunoblots of LC3 was expressed as the ratio of LC3-II to LC3-I, and the ratio of LC3II/LC3I was expressed in arbitrary units. (C) Western blot visualized LC3-I and LC3-II protein levels in starvation (EBSS medium containing 1mM pepstatin A and E-64-d solution) treated Caco2 cells. The ratio of LC3II/LC3I was expressed in arbitrary units. (D) Starvation significantly inhibited rotavirus RNA in Caco2 cells (n = 6, mean ± SEM, *P

    Journal: Virulence

    Article Title: PI3K-Akt-mTOR axis sustains rotavirus infection via the 4E-BP1 mediated autophagy pathway and represents an antiviral target

    doi: 10.1080/21505594.2017.1326443

    Figure Lengend Snippet: 4E-BP1 mediates rapamycin-induced autophagy that inhibits rotavirus infection. (A) Caco2 cells transduced with lentiviral particles carrying a construct of TagGFP2-LC3 driven by the elongation factor-1 promotor were cultured at 37°C for 48 h in DMEM medium containing EBSS medium containing 1mM pepstatin A and E-64-d solution (for starvation) and 10 nM rapamycin. LC3-positive puncta was observed by confocal laser microscopy. (B) LC3-I and LC3-II protein levels were examined by western blot analysis. Protein samples were extracted from Caco2 cells treated with indicated concentrations of rapamycin (48 h). Quantification of the intensity of the immunoreactive bands of both LC3-I and LC3-II was performed using Odyssey V3.0 software. Densitometric analysis of immunoblots of LC3 was expressed as the ratio of LC3-II to LC3-I, and the ratio of LC3II/LC3I was expressed in arbitrary units. (C) Western blot visualized LC3-I and LC3-II protein levels in starvation (EBSS medium containing 1mM pepstatin A and E-64-d solution) treated Caco2 cells. The ratio of LC3II/LC3I was expressed in arbitrary units. (D) Starvation significantly inhibited rotavirus RNA in Caco2 cells (n = 6, mean ± SEM, *P

    Article Snippet: Other reagents including Earle's Balanced Salt Solution (EBSS) (Lonza), E-64-d (Santa Cruz Biotech, Santa Cruz, CA) and pepstatin A (Santa Cruz Biotech, Santa Cruz, CA) were also used.

    Techniques: Infection, Transduction, Construct, Cell Culture, Microscopy, Western Blot, Software

    Inhibition of autophagy rescues cell death in Mar-treated PC3 cells. The cell death of PC3 cells exposed to Mar alone or combined treatment. Left panel, cell death by PI exclusion. Right panel, immunoblot analysis of LC3BII, Atg5 and p62 levels in PC3 cells after treatments. OD of LC3BII relative to the control was quantified as mean±S.D., n =3. ( a ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 5 mM 3-MA for 24 h. ( b ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 10 μ M E-64D/10 μ M Pepstatin A (Pep) for 24 h. ( c ) Knockdown of Atg5 by siRNA for 24 or 48 h in PC3 cells and then treated with 10 μ M Mar. ( d ) Knockdown of LC3B by siRNA for 24 or 48 h in PC3 cells prior to Mar treatment. ** P

    Journal: Cell Death & Disease

    Article Title: Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells

    doi: 10.1038/cddis.2013.285

    Figure Lengend Snippet: Inhibition of autophagy rescues cell death in Mar-treated PC3 cells. The cell death of PC3 cells exposed to Mar alone or combined treatment. Left panel, cell death by PI exclusion. Right panel, immunoblot analysis of LC3BII, Atg5 and p62 levels in PC3 cells after treatments. OD of LC3BII relative to the control was quantified as mean±S.D., n =3. ( a ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 5 mM 3-MA for 24 h. ( b ) PC3 cells treated with 10 μ M Mar or 10 μ M Mar plus 10 μ M E-64D/10 μ M Pepstatin A (Pep) for 24 h. ( c ) Knockdown of Atg5 by siRNA for 24 or 48 h in PC3 cells and then treated with 10 μ M Mar. ( d ) Knockdown of LC3B by siRNA for 24 or 48 h in PC3 cells prior to Mar treatment. ** P

    Article Snippet: The purified 20S proteasome, z-VAD-fmk, E-64D, Pepstatin A, N -succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC), benzyloxycarbonyl-L -leucyl-leucyl-glutamyl-methylcoumarylamide (Bz-LLE-AMC) and Bz-Val-Gly-Arg-7-amino-4-methylcoumarin (Bz-VGR-AMC) were purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA).

    Techniques: Inhibition

    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

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

    Journal: The FASEB Journal

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

    doi: 10.1096/fj.13-236166

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

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

    Techniques: Activation Assay, Binding Assay

    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: In MTX-treated rats receiving pepstatin A or vehicle, villus height decreased compared with control rats ( P < 0·05, ).

    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: In MTX-treated rats receiving pepstatin A or vehicle, villus height decreased compared with control rats ( P < 0·05, ).

    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: In MTX-treated rats receiving pepstatin A or vehicle, villus height decreased compared with control rats ( P < 0·05, ).

    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: Acetyl pepstatin was selected as the weak inhibitor in the displacement titrations because this inhibitor is endothermic and amplifies the signal of a high-affinity exothermic inhibitor when displaced.

    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: Acetyl pepstatin was selected as the weak inhibitor in the displacement titrations because this inhibitor is endothermic and amplifies the signal of a high-affinity exothermic inhibitor when displaced.

    Techniques: Titration, Injection

    EWS-Fli-1 turns over by a lysosome-dependent mechanism: (A) Knockdown of CIMPR or VPS26A results in destabilization of FLAG-EWS-Fli-1. 293 cells were cotransfected with FLAG-EWS-Fli-1 and shRNA against luciferase (control), CIMPR, or VPS26A. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (B) TFEB induces EWS-Fli-1 degradation in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and HA-TFEB or empty vector. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (C) Chloroquine stabilizes EWS-Fli-1 in 293 cells. 293 cells were transfected with FLAG-EWS-Fli-1. Transfected cells were left untreated (control) or treated with 100 μM chloroquine for 12 h. The levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (D) Cathepsin D degrades EWS-Fli-1, but not p53, in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and cathepsin D or empty vector. Transfected cells were left untreated or treated with 100 μM chloroquine for 12 h or 100 nM pepstatin A for 12 h. 293 cells were cotransfected with FLAG-p53 and cathepsin D or empty vector. The levels of FLAG-EWS-Fli-1 and FLAG-p53 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (E) Cathepsin D degrades endogenous EWS-Fli-1 in A673 Ewing sarcoma cells. A673 cells were infected with a lentivirus vector expressing cathepsin D or an empty vector, the infected cells were selected with puromycin, and the levels of endogenous EWS-Fli-1 were examined by anti-Fli-1 C-terminus antibody immunoblotting at 4 days after infection. Nucleolin serves as a loading control. (F) Chloroquine stabilizes endogenous EWS-Fli-1 in A673 cells. A673 cells were left untreated, treated with 100 μM chloroquine for 12 h, or treated with 10 μM MG-132 for 12 h. The levels of EWS-Fli-1 were examined by anti-Fli-1 C-terminus immunoblotting. While chloroquine increased the levels of endogenous EWS-Fli-1, MG-132 had no effect on the EWS-Fli-1 protein levels, suggesting that EWS-Fli-1 turns over by a lysosomal, but not proteasomal mechanism. (G) Endogenous EWS-Fli-1 in A673 cells displays increased lysosomal location upon chloroquine treatment. A673 cells were treated with 100 μM chloroquine for 12 h or left untreated, and the whole cell extract (WCE) and lysosomal fraction were isolated. The abundance of EWS-Fli-1, LAMP2, p62/SQSTM1, and mSin3A in each fraction was determined by immunoblotting.

    Journal: Journal of Proteome Research

    Article Title: Proteomic Analysis of the EWS-Fli-1 Interactome Reveals the Role of the Lysosome in EWS-Fli-1 Turnover

    doi: 10.1021/pr500387m

    Figure Lengend Snippet: EWS-Fli-1 turns over by a lysosome-dependent mechanism: (A) Knockdown of CIMPR or VPS26A results in destabilization of FLAG-EWS-Fli-1. 293 cells were cotransfected with FLAG-EWS-Fli-1 and shRNA against luciferase (control), CIMPR, or VPS26A. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (B) TFEB induces EWS-Fli-1 degradation in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and HA-TFEB or empty vector. Forty-eight hours after transfection, the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (C) Chloroquine stabilizes EWS-Fli-1 in 293 cells. 293 cells were transfected with FLAG-EWS-Fli-1. Transfected cells were left untreated (control) or treated with 100 μM chloroquine for 12 h. The levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting. Tubulin serves as a loading control. (D) Cathepsin D degrades EWS-Fli-1, but not p53, in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1 and cathepsin D or empty vector. Transfected cells were left untreated or treated with 100 μM chloroquine for 12 h or 100 nM pepstatin A for 12 h. 293 cells were cotransfected with FLAG-p53 and cathepsin D or empty vector. The levels of FLAG-EWS-Fli-1 and FLAG-p53 were examined by anti-FLAG immunoblotting. Nucleolin serves as a loading control. (E) Cathepsin D degrades endogenous EWS-Fli-1 in A673 Ewing sarcoma cells. A673 cells were infected with a lentivirus vector expressing cathepsin D or an empty vector, the infected cells were selected with puromycin, and the levels of endogenous EWS-Fli-1 were examined by anti-Fli-1 C-terminus antibody immunoblotting at 4 days after infection. Nucleolin serves as a loading control. (F) Chloroquine stabilizes endogenous EWS-Fli-1 in A673 cells. A673 cells were left untreated, treated with 100 μM chloroquine for 12 h, or treated with 10 μM MG-132 for 12 h. The levels of EWS-Fli-1 were examined by anti-Fli-1 C-terminus immunoblotting. While chloroquine increased the levels of endogenous EWS-Fli-1, MG-132 had no effect on the EWS-Fli-1 protein levels, suggesting that EWS-Fli-1 turns over by a lysosomal, but not proteasomal mechanism. (G) Endogenous EWS-Fli-1 in A673 cells displays increased lysosomal location upon chloroquine treatment. A673 cells were treated with 100 μM chloroquine for 12 h or left untreated, and the whole cell extract (WCE) and lysosomal fraction were isolated. The abundance of EWS-Fli-1, LAMP2, p62/SQSTM1, and mSin3A in each fraction was determined by immunoblotting.

    Article Snippet: Reagents Chloroquine and pepstatin A were purchased from MP Biomedicals.

    Techniques: shRNA, Luciferase, Transfection, Plasmid Preparation, Infection, Expressing, Isolation

    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