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  • 90
    Thermo Fisher leupeptin
    The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; <t>Leupeptin,</t> 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P
    Leupeptin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1696 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore leupeptin
    The decrease of NIS protein levels by MEK inhibition is mediated by lysosomal but not proteosomal pathway in MCF-7-tRA/H cells A. Western blot analysis showed that proteasome inhibitors did not prevent the decrease of NIS protein levels in U0126 treated MCF-7-tRA/H cells. MCF-7 cells treated with tRA/H were cultured in the presence of DMSO or U0126 for 24 hours, along with or without proteasome inhibitors MG132 or β-lactone for 8 hours. β-actin served as a loading control and p53 was included to ensure the efficacy of proteasome inhibition. The results are representative of two independent experiments. B. Bar graph indicates the densitometry values of NIS protein level normalized with β-actin of cells under various treatments (represented as percentage relative to MCF-7/tRA/H). C. Western blot analysis showed that lysosome inhibitors prevented the decrease of NIS protein levels in U0126 treated MCF-7-tRA/H cells. MCF-7 cells treated with tRA/H were cultured in the presence of DMSO or U0126 for 24 hours, along with or without lysosome inhibitors <t>leupeptin</t> or chloroquine for 8 hours. β-actin served as a loading control. The results are representative of three independent experiments. D. Bar graph indicates the densitometry values of NIS protein level normalized with β-actin of cells under various treatments (represented as percentage relative to MCF-7/tRA/H).
    Leupeptin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Selleck Chemicals leupeptin
    The decrease of NIS protein levels by MEK inhibition is mediated by lysosomal but not proteosomal pathway in MCF-7-tRA/H cells A. Western blot analysis showed that proteasome inhibitors did not prevent the decrease of NIS protein levels in U0126 treated MCF-7-tRA/H cells. MCF-7 cells treated with tRA/H were cultured in the presence of DMSO or U0126 for 24 hours, along with or without proteasome inhibitors MG132 or β-lactone for 8 hours. β-actin served as a loading control and p53 was included to ensure the efficacy of proteasome inhibition. The results are representative of two independent experiments. B. Bar graph indicates the densitometry values of NIS protein level normalized with β-actin of cells under various treatments (represented as percentage relative to MCF-7/tRA/H). C. Western blot analysis showed that lysosome inhibitors prevented the decrease of NIS protein levels in U0126 treated MCF-7-tRA/H cells. MCF-7 cells treated with tRA/H were cultured in the presence of DMSO or U0126 for 24 hours, along with or without lysosome inhibitors <t>leupeptin</t> or chloroquine for 8 hours. β-actin served as a loading control. The results are representative of three independent experiments. D. Bar graph indicates the densitometry values of NIS protein level normalized with β-actin of cells under various treatments (represented as percentage relative to MCF-7/tRA/H).
    Leupeptin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Tocris leupeptin
    Prolonged sodium pervanadate treatment induces removal of KCC2 from the cell surface and results in KCC2 degradation. A. Cultured hippocampal neurons were treated with 200 µg/ml <t>leupeptin</t> or 80 µM dynasore for 45 min. The amount of surface
    Leupeptin, supplied by Tocris, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Adipogen leupeptin
    Prolonged sodium pervanadate treatment induces removal of KCC2 from the cell surface and results in KCC2 degradation. A. Cultured hippocampal neurons were treated with 200 µg/ml <t>leupeptin</t> or 80 µM dynasore for 45 min. The amount of surface
    Leupeptin, supplied by Adipogen, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime leupeptin
    Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or <t>leupeptin</t> (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p
    Leupeptin, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio Basic Canada leupeptin
    Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or <t>leupeptin</t> (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p
    Leupeptin, supplied by Bio Basic Canada, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad leupeptin
    Short form STAT5 occurs naturally in prostate cancer cells A , Electromobility shift assay of nuclear extract of DU145 cells prepared from flash frozen cell pellets in the absence (lane 1 and 2) or presence (lane 3 and 4) of protease inhibitors (phenylmethylsulfonylfluoride, aprotinin, <t>leupeptin</t> and pepstatin-A). The short form Stat5a/b (arrow) was detected in DU145 cells in the presence or in the absence of protease inhibitors, and the short form Stat5a/b was super-shifted by anti-Stat5a and anti-Stat5b antibodies. B , The short form Stat5a/b is not generated in vitro after disruption of the cell membranes. DU145 cell pellets were flash frozen in liquid nitrogen and directly lysed in boiling SDS loading buffer (lane 1), or the cell pellets were lysed in cell lysis buffer in the presence (lane 2) or in the absence of proteases inhibitors (lane 3) and boiled in SDS loading buffer and analyzed by Western blotting using anti-C-terminus Stat5a/b mAb.
    Leupeptin, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 633 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioShop leupeptin
    Rac1 promotes Rab5-dependent endocytosis of Crb. (A-E) Embryos were co-stained for Crb (red in the merged images) and Arm (green). Panels depict a surface view of the ventral ectoderm of the following embryos: Control (A: da-GAL4 embryo incubated in DMSO), Rac1 V12 (B: da-GAL4/UAS-Rac1 V12 embryo incubated in DMSO), Rac1 V12 <t>Leupeptin</t> (C: da-GAL4/UAS-Rac1 V12 embryo treated with the lysosomal proteolysis inhibitor leupeptin), Rac1 V12 Dynasore (D: da-GAL4/UAS-Rac1 V12 embryo treated with the dynamin inhibitor dynasore), Rac1 V12 Rab5 S43N ( da-GAL4/UAS-Rac1 V12 , UAS-Rab5 S43N ). Images are representative results of three independent experiments. Scale bar: 10 µm. C, arrows point to intracellular puncta; D,E, arrows indicate cell-cell borders. (F-H) Stage 16 Rab5 S43N -expressing embryos stained for 2A12 (F), Serp (G) or Verm (H). (I) 2A12 staining performed on an embryo expressing Rab5 S43N in the trachea and heterozygous for the null allele crb 11A22 ( btl-GAL4; UAS-Rab5 S43N / crb 11A22 ). Scale bar: 100 µm. (J) Histogram showing the quantification of the dorsal trunk length in Rab5 S43N ( btl-GAL4; UAS-Rab5 S43N ) and Rab5 S43N , crb /+ ( btl-GAL4; UAS-Rab5 S43N , crb 11A22 /+ ) embryos relative to dorsal trunk length of control animals ( btl-GAL4 ). n =15 embryos for each genotype, taken from at least three independent experiments. Bars represent mean±s.d. A two-tailed t -test was used to evaluate the statistical significance; ** P
    Leupeptin, supplied by BioShop, used in various techniques. Bioz Stars score: 93/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim leupeptin
    Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml <t>leupeptin</t> (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).
    Leupeptin, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 778 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc leupeptin
    Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml <t>leupeptin</t> (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).
    Leupeptin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1060 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific leupeptin
    In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of <t>leupeptin</t> (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P
    Leupeptin, supplied by Fisher Scientific, 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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    GE Healthcare leupeptin
    Cidea is a short-lived protein and its degradation is proteasome-dependent ( A , B and C ) A 1 μg portion of CMV5–HA–hCidea was co-transfected with 0.5 μg of pEGFP-N1 into HEK-293T, CHO-K1 and H1299 cells in 60-mm-diameter dishes using Dosper reagent. CHX-based protein chase experiments were performed as described below. At 24 h post-transfection, and 1 h prior to the addition of CHX, the medium was replaced with fresh medium, and then CHX was added to a final concentration of 100 μg/ml. Cells were harvested in 0.5 ml of lysis buffer at different time points: 0, 30, 60 and 120 min. Total cell lysates were prepared and analysed by Western blotting [IB (immunoblotting)] using anti-HA and anti-GFP as primary antibodies. The amount of GFP transiently co-expressed by pEGFP-N1 present in total cell lysates was used for the normalization of transfection protein. ( D ) Graph showing band intensities corresponding to HA–Cidea and GFP scanned and quantitified using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. ( E ) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV-HA vector (lane 1) or 1 μg of CMV5–HA–hCidea (lane 2–11) and 0.5 μg of pEGFP-N1 using the calcium phosphate method. After 24 h, CHX was added to 100 μg/ml (lanes 3–11) along with different reagents [DMSO, ethanol, pepstatin (0.5 μg/ml), <t>leupeptin</t> (5 μg/ml), aprotinin (2 μg/ml), ALLN (50 μM), MG132 (10 μM), chloroquine (50 μg/ml) and NH 4 Cl (2.5 mM)] for 2 h. Cells were harvested in 0.5 ml of lysis buffer after the 2 h treatment. Total cell lysates were subjected to Western blotting and detected with anti-HA and anti-GFP antibodies.
    Leupeptin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ICN Biomedicals leupeptin
    Cidea is a short-lived protein and its degradation is proteasome-dependent ( A , B and C ) A 1 μg portion of CMV5–HA–hCidea was co-transfected with 0.5 μg of pEGFP-N1 into HEK-293T, CHO-K1 and H1299 cells in 60-mm-diameter dishes using Dosper reagent. CHX-based protein chase experiments were performed as described below. At 24 h post-transfection, and 1 h prior to the addition of CHX, the medium was replaced with fresh medium, and then CHX was added to a final concentration of 100 μg/ml. Cells were harvested in 0.5 ml of lysis buffer at different time points: 0, 30, 60 and 120 min. Total cell lysates were prepared and analysed by Western blotting [IB (immunoblotting)] using anti-HA and anti-GFP as primary antibodies. The amount of GFP transiently co-expressed by pEGFP-N1 present in total cell lysates was used for the normalization of transfection protein. ( D ) Graph showing band intensities corresponding to HA–Cidea and GFP scanned and quantitified using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. ( E ) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV-HA vector (lane 1) or 1 μg of CMV5–HA–hCidea (lane 2–11) and 0.5 μg of pEGFP-N1 using the calcium phosphate method. After 24 h, CHX was added to 100 μg/ml (lanes 3–11) along with different reagents [DMSO, ethanol, pepstatin (0.5 μg/ml), <t>leupeptin</t> (5 μg/ml), aprotinin (2 μg/ml), ALLN (50 μM), MG132 (10 μM), chloroquine (50 μg/ml) and NH 4 Cl (2.5 mM)] for 2 h. Cells were harvested in 0.5 ml of lysis buffer after the 2 h treatment. Total cell lysates were subjected to Western blotting and detected with anti-HA and anti-GFP antibodies.
    Leupeptin, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 92/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Peptide Institute leupeptin
    Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM <t>leupeptin</t> with or without 100 nM epoxomycin, according to the same protocol as in A.
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    Peptides International leupeptin
    Propranolol (PPL) modulates ß-AR degradation in human dermal fibroblasts Twenty-four hours after being plated, NF, HSF, and NSF fibroblasts were pretreated for 30 minutes with PPL or vehicle and then treated for the indicated time with a proteasome inhibitor (MG132) (10 μM) or lysosomal inhibitor <t>(leupeptin)</t> (50 μM). Western blotting was performed to determine the expression of ß1-, ß2-, and ß3-ARs in the presence of a. vehicle or b. PPL (10 μM). c. Quantification of ß1-AR band intensity. d. Quantification of ß2-AR band intensity. e. Quantification of ß3-AR band intensity. Data were normalized to control and were presented as mean ± SEM of at least 3 independent experiments. *p
    Leupeptin, supplied by Peptides International, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche leupeptin
    Schematic representation of aSMase processing. A , pro-aSMase (72–75 kDa) arises from prepro-aSMase (75 kDa) within the Golgi and is either targeted to the lysosome via mannose 6-phosphorylation ( M6P ), or is released extracellularly through the default Golgi secretory pathway giving rise to S-SMase (75–80 kDa). Upon entry to the endolysosomal compartment pro-aSMase is cleaved by “protease X” giving rise to the 65-kDa aSMase, which upon encountering Zn 2+ in the lysosome becomes L-SMase. L-SMase (65 kDa) is in turn degraded by “protease Y” to the 52-kDa inactive L-SMase. The action of protease Y is promoted by desipramine, and inhibited by <t>leupeptin.</t> B , both pro-aSMase and S-SMase have an intact C terminus, require exogenous Zn 2+ for activity, and are insensitive to desipramine. Mature L-SMase has lost a portion of its C terminus, which may promote coordination of lysosomal Zn 2+ , thereby rendering the 65-kDa aSMase, Zn 2+ -independent, and is sensitive to desipramine.
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    Santa Cruz Biotechnology leupeptin
    Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with <t>leupeptin</t> (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P
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    Applichem leupeptin
    Inhibitory analysis of L1 proteolysis. HeLa (A) or HaCaT (B) cells were infected with HPV16 PsV in the presence or absence of the following inhibitors: NH 4 Cl (20 mM), bafilomycin A1 (10 nM), E-64 (100 μM), <t>leupeptin</t> (100 μM), CA-074 or
    Leupeptin, supplied by Applichem, used in various techniques. Bioz Stars score: 93/100, based on 139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH leupeptin
    Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor <t>(leupeptin),</t> 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p
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    Abrogation of pro-HGF mediated c-met activation and breast cancer cell proliferation using matriptase inhibitors (a) Matriptase sufficient mouse mammary carcinoma cells were isolated from Mat-PymT + mice and exposed to pro-HGF or pre-cleaved active HGF with and without matriptase inhibitors added concomitantly (60 nM HAI-1, 40 nM HAI-2 or 20 uM <t>leupeptin).</t> Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (b) Mouse mammary carcinoma cells or human breast cancer BT20 cells were serum starved and pre-incubated with 1 μM IN-1 synthetic matriptase inhibitor for 30 min before exposure to active HGF, pro-HGF, or no growth factors. Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (c) BT20 cells were serum starved and exposed to either active HGF (gray bars), pro-HGF (white bars), or no growth factor (black bars) for 24 h and counted. The synthetic selective matriptase inhibitor IN-1 was added at 10 nM, 100 nM, 1 μM, or 10 μM concomitantly with pro-HGF/HGF. In the mock control vehicle (PBS) was added. Graphs show the increase in cell numbers at 24 h after stimulation (triplicates) (*P
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    Sangon Biotech leupeptin
    Abrogation of pro-HGF mediated c-met activation and breast cancer cell proliferation using matriptase inhibitors (a) Matriptase sufficient mouse mammary carcinoma cells were isolated from Mat-PymT + mice and exposed to pro-HGF or pre-cleaved active HGF with and without matriptase inhibitors added concomitantly (60 nM HAI-1, 40 nM HAI-2 or 20 uM <t>leupeptin).</t> Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (b) Mouse mammary carcinoma cells or human breast cancer BT20 cells were serum starved and pre-incubated with 1 μM IN-1 synthetic matriptase inhibitor for 30 min before exposure to active HGF, pro-HGF, or no growth factors. Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (c) BT20 cells were serum starved and exposed to either active HGF (gray bars), pro-HGF (white bars), or no growth factor (black bars) for 24 h and counted. The synthetic selective matriptase inhibitor IN-1 was added at 10 nM, 100 nM, 1 μM, or 10 μM concomitantly with pro-HGF/HGF. In the mock control vehicle (PBS) was added. Graphs show the increase in cell numbers at 24 h after stimulation (triplicates) (*P
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    Abrogation of pro-HGF mediated c-met activation and breast cancer cell proliferation using matriptase inhibitors (a) Matriptase sufficient mouse mammary carcinoma cells were isolated from Mat-PymT + mice and exposed to pro-HGF or pre-cleaved active HGF with and without matriptase inhibitors added concomitantly (60 nM HAI-1, 40 nM HAI-2 or 20 uM <t>leupeptin).</t> Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (b) Mouse mammary carcinoma cells or human breast cancer BT20 cells were serum starved and pre-incubated with 1 μM IN-1 synthetic matriptase inhibitor for 30 min before exposure to active HGF, pro-HGF, or no growth factors. Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (c) BT20 cells were serum starved and exposed to either active HGF (gray bars), pro-HGF (white bars), or no growth factor (black bars) for 24 h and counted. The synthetic selective matriptase inhibitor IN-1 was added at 10 nM, 100 nM, 1 μM, or 10 μM concomitantly with pro-HGF/HGF. In the mock control vehicle (PBS) was added. Graphs show the increase in cell numbers at 24 h after stimulation (triplicates) (*P
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    Abrogation of pro-HGF mediated c-met activation and breast cancer cell proliferation using matriptase inhibitors (a) Matriptase sufficient mouse mammary carcinoma cells were isolated from Mat-PymT + mice and exposed to pro-HGF or pre-cleaved active HGF with and without matriptase inhibitors added concomitantly (60 nM HAI-1, 40 nM HAI-2 or 20 uM <t>leupeptin).</t> Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (b) Mouse mammary carcinoma cells or human breast cancer BT20 cells were serum starved and pre-incubated with 1 μM IN-1 synthetic matriptase inhibitor for 30 min before exposure to active HGF, pro-HGF, or no growth factors. Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (c) BT20 cells were serum starved and exposed to either active HGF (gray bars), pro-HGF (white bars), or no growth factor (black bars) for 24 h and counted. The synthetic selective matriptase inhibitor IN-1 was added at 10 nM, 100 nM, 1 μM, or 10 μM concomitantly with pro-HGF/HGF. In the mock control vehicle (PBS) was added. Graphs show the increase in cell numbers at 24 h after stimulation (triplicates) (*P
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    Evaluation of the overall performance of the nanofractionation system using the plasmin inhibitor <t>leupeptin,</t> which was nanofractionated at five concentrations injected. ( a ) 6.3 μM. ( b ) 25 μM. ( c ) 50 μM. ( d ) 100 μM. ( e ) 200 μM. ( f ) The extracted ion chromatogram ( m/z 427.3, [M + H] + ) of the analysis of 100 μM leupeptin with online LC-MS. RFU, relative fluorescence units
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    Image Search Results


    The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

    Journal: Scientific Reports

    Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity

    doi: 10.1038/s41598-018-31364-y

    Figure Lengend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P

    Article Snippet: Briefly, cervical cancer cells were co-transfected with plasmids containing NaV 1.6 and GFP genes and seeded in the inserts at 1 × 105 cells density using culture medium with 5% FBS in absence or presence of 1-µM TTX, or in presence of protease inhibitors: GM6001, 25 µM (Millipore); E-64, 100 µM (Calbiochem; San Diego, CA); Leupeptin, 100 µM (Thermo Fisher Scientific), or the NHE-1 specific inhibitor 5-( N -ethyl- N -isopropyl) amiloride, EIPA, 1 µM (Sigma-Aldrich; St. Louis, MO).

    Techniques: Activity Assay, Transfection, Staining

    The decrease of NIS protein levels by MEK inhibition is mediated by lysosomal but not proteosomal pathway in MCF-7-tRA/H cells A. Western blot analysis showed that proteasome inhibitors did not prevent the decrease of NIS protein levels in U0126 treated MCF-7-tRA/H cells. MCF-7 cells treated with tRA/H were cultured in the presence of DMSO or U0126 for 24 hours, along with or without proteasome inhibitors MG132 or β-lactone for 8 hours. β-actin served as a loading control and p53 was included to ensure the efficacy of proteasome inhibition. The results are representative of two independent experiments. B. Bar graph indicates the densitometry values of NIS protein level normalized with β-actin of cells under various treatments (represented as percentage relative to MCF-7/tRA/H). C. Western blot analysis showed that lysosome inhibitors prevented the decrease of NIS protein levels in U0126 treated MCF-7-tRA/H cells. MCF-7 cells treated with tRA/H were cultured in the presence of DMSO or U0126 for 24 hours, along with or without lysosome inhibitors leupeptin or chloroquine for 8 hours. β-actin served as a loading control. The results are representative of three independent experiments. D. Bar graph indicates the densitometry values of NIS protein level normalized with β-actin of cells under various treatments (represented as percentage relative to MCF-7/tRA/H).

    Journal: Endocrine-related cancer

    Article Title: MEK Inhibition Leads To Lysosome-Mediated Na+/I- Symporter Protein Degradation In Human Breast Cancer Cells

    doi: 10.1530/ERC-12-0342

    Figure Lengend Snippet: The decrease of NIS protein levels by MEK inhibition is mediated by lysosomal but not proteosomal pathway in MCF-7-tRA/H cells A. Western blot analysis showed that proteasome inhibitors did not prevent the decrease of NIS protein levels in U0126 treated MCF-7-tRA/H cells. MCF-7 cells treated with tRA/H were cultured in the presence of DMSO or U0126 for 24 hours, along with or without proteasome inhibitors MG132 or β-lactone for 8 hours. β-actin served as a loading control and p53 was included to ensure the efficacy of proteasome inhibition. The results are representative of two independent experiments. B. Bar graph indicates the densitometry values of NIS protein level normalized with β-actin of cells under various treatments (represented as percentage relative to MCF-7/tRA/H). C. Western blot analysis showed that lysosome inhibitors prevented the decrease of NIS protein levels in U0126 treated MCF-7-tRA/H cells. MCF-7 cells treated with tRA/H were cultured in the presence of DMSO or U0126 for 24 hours, along with or without lysosome inhibitors leupeptin or chloroquine for 8 hours. β-actin served as a loading control. The results are representative of three independent experiments. D. Bar graph indicates the densitometry values of NIS protein level normalized with β-actin of cells under various treatments (represented as percentage relative to MCF-7/tRA/H).

    Article Snippet: Leupeptin and Chloroquine (Sigma) were dissolved in distilled water at 10mM.

    Techniques: Inhibition, Western Blot, Cell Culture

    Prolonged sodium pervanadate treatment induces removal of KCC2 from the cell surface and results in KCC2 degradation. A. Cultured hippocampal neurons were treated with 200 µg/ml leupeptin or 80 µM dynasore for 45 min. The amount of surface

    Journal: Molecular and cellular neurosciences

    Article Title: Tyrosine phosphorylation regulates the membrane trafficking of the potassium chloride co-transporter KCC2

    doi: 10.1016/j.mcn.2010.06.008

    Figure Lengend Snippet: Prolonged sodium pervanadate treatment induces removal of KCC2 from the cell surface and results in KCC2 degradation. A. Cultured hippocampal neurons were treated with 200 µg/ml leupeptin or 80 µM dynasore for 45 min. The amount of surface

    Article Snippet: Leupeptin (Tocris) was diluted in water to make a 10 mg/ml stock solution.

    Techniques: Cell Culture

    Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p

    Journal: Scientific Reports

    Article Title: EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells

    doi: 10.1038/srep11009

    Figure Lengend Snippet: Different degradation pathway regulated the inverse interaction between topoisomerase IIα and JWA protein expression. ( a ) and ( b ) NCI-H460 cells were transiently transfected with Flag-JWA plasmid (2.5 μg) for 24 h, incubated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. Western blotting was carried out to confirm the level of topoisomerase IIα. ( c ) and (d) NCI-H460 cells were transiently transfected with Flag- topoisomerase IIα plasmid (2.5 μg) for 24 h and co-treated with (+) or without (-) MG-132 (5 μM) for 8 h or leupeptin (5 μM) for 20 h. JWA protein was detected by anti-JWA antibody. β-actin expression served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p

    Article Snippet: Nocodazole, MG132 and Leupeptin were obtained from Beyotime Institute of Biotechnology (Shanghai, China).

    Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot

    Short form STAT5 occurs naturally in prostate cancer cells A , Electromobility shift assay of nuclear extract of DU145 cells prepared from flash frozen cell pellets in the absence (lane 1 and 2) or presence (lane 3 and 4) of protease inhibitors (phenylmethylsulfonylfluoride, aprotinin, leupeptin and pepstatin-A). The short form Stat5a/b (arrow) was detected in DU145 cells in the presence or in the absence of protease inhibitors, and the short form Stat5a/b was super-shifted by anti-Stat5a and anti-Stat5b antibodies. B , The short form Stat5a/b is not generated in vitro after disruption of the cell membranes. DU145 cell pellets were flash frozen in liquid nitrogen and directly lysed in boiling SDS loading buffer (lane 1), or the cell pellets were lysed in cell lysis buffer in the presence (lane 2) or in the absence of proteases inhibitors (lane 3) and boiled in SDS loading buffer and analyzed by Western blotting using anti-C-terminus Stat5a/b mAb.

    Journal: The international journal of biochemistry & cell biology

    Article Title: N-Terminal Truncation of Stat5a/b Circumvents PIAS3-Mediated Transcriptional Inhibition of Stat5 in Prostate Cancer Cells

    doi: 10.1016/j.biocel.2010.09.008

    Figure Lengend Snippet: Short form STAT5 occurs naturally in prostate cancer cells A , Electromobility shift assay of nuclear extract of DU145 cells prepared from flash frozen cell pellets in the absence (lane 1 and 2) or presence (lane 3 and 4) of protease inhibitors (phenylmethylsulfonylfluoride, aprotinin, leupeptin and pepstatin-A). The short form Stat5a/b (arrow) was detected in DU145 cells in the presence or in the absence of protease inhibitors, and the short form Stat5a/b was super-shifted by anti-Stat5a and anti-Stat5b antibodies. B , The short form Stat5a/b is not generated in vitro after disruption of the cell membranes. DU145 cell pellets were flash frozen in liquid nitrogen and directly lysed in boiling SDS loading buffer (lane 1), or the cell pellets were lysed in cell lysis buffer in the presence (lane 2) or in the absence of proteases inhibitors (lane 3) and boiled in SDS loading buffer and analyzed by Western blotting using anti-C-terminus Stat5a/b mAb.

    Article Snippet: Cells were lysed and fresh human prostate cancer specimens were homogenized in lysis buffer [10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulphonylfluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin], and the protein concentrations of the whole cell lysates were determined by the Bradford method (BioRad Laboratories Inc., Hercules, CA).

    Techniques: Electro Mobility Shift Assay, Generated, In Vitro, Lysis, Western Blot

    Rac1 promotes Rab5-dependent endocytosis of Crb. (A-E) Embryos were co-stained for Crb (red in the merged images) and Arm (green). Panels depict a surface view of the ventral ectoderm of the following embryos: Control (A: da-GAL4 embryo incubated in DMSO), Rac1 V12 (B: da-GAL4/UAS-Rac1 V12 embryo incubated in DMSO), Rac1 V12 Leupeptin (C: da-GAL4/UAS-Rac1 V12 embryo treated with the lysosomal proteolysis inhibitor leupeptin), Rac1 V12 Dynasore (D: da-GAL4/UAS-Rac1 V12 embryo treated with the dynamin inhibitor dynasore), Rac1 V12 Rab5 S43N ( da-GAL4/UAS-Rac1 V12 , UAS-Rab5 S43N ). Images are representative results of three independent experiments. Scale bar: 10 µm. C, arrows point to intracellular puncta; D,E, arrows indicate cell-cell borders. (F-H) Stage 16 Rab5 S43N -expressing embryos stained for 2A12 (F), Serp (G) or Verm (H). (I) 2A12 staining performed on an embryo expressing Rab5 S43N in the trachea and heterozygous for the null allele crb 11A22 ( btl-GAL4; UAS-Rab5 S43N / crb 11A22 ). Scale bar: 100 µm. (J) Histogram showing the quantification of the dorsal trunk length in Rab5 S43N ( btl-GAL4; UAS-Rab5 S43N ) and Rab5 S43N , crb /+ ( btl-GAL4; UAS-Rab5 S43N , crb 11A22 /+ ) embryos relative to dorsal trunk length of control animals ( btl-GAL4 ). n =15 embryos for each genotype, taken from at least three independent experiments. Bars represent mean±s.d. A two-tailed t -test was used to evaluate the statistical significance; ** P

    Journal: Biology Open

    Article Title: Rac1 controls epithelial tube length through the apical secretion and polarity pathways

    doi: 10.1242/bio.015727

    Figure Lengend Snippet: Rac1 promotes Rab5-dependent endocytosis of Crb. (A-E) Embryos were co-stained for Crb (red in the merged images) and Arm (green). Panels depict a surface view of the ventral ectoderm of the following embryos: Control (A: da-GAL4 embryo incubated in DMSO), Rac1 V12 (B: da-GAL4/UAS-Rac1 V12 embryo incubated in DMSO), Rac1 V12 Leupeptin (C: da-GAL4/UAS-Rac1 V12 embryo treated with the lysosomal proteolysis inhibitor leupeptin), Rac1 V12 Dynasore (D: da-GAL4/UAS-Rac1 V12 embryo treated with the dynamin inhibitor dynasore), Rac1 V12 Rab5 S43N ( da-GAL4/UAS-Rac1 V12 , UAS-Rab5 S43N ). Images are representative results of three independent experiments. Scale bar: 10 µm. C, arrows point to intracellular puncta; D,E, arrows indicate cell-cell borders. (F-H) Stage 16 Rab5 S43N -expressing embryos stained for 2A12 (F), Serp (G) or Verm (H). (I) 2A12 staining performed on an embryo expressing Rab5 S43N in the trachea and heterozygous for the null allele crb 11A22 ( btl-GAL4; UAS-Rab5 S43N / crb 11A22 ). Scale bar: 100 µm. (J) Histogram showing the quantification of the dorsal trunk length in Rab5 S43N ( btl-GAL4; UAS-Rab5 S43N ) and Rab5 S43N , crb /+ ( btl-GAL4; UAS-Rab5 S43N , crb 11A22 /+ ) embryos relative to dorsal trunk length of control animals ( btl-GAL4 ). n =15 embryos for each genotype, taken from at least three independent experiments. Bars represent mean±s.d. A two-tailed t -test was used to evaluate the statistical significance; ** P

    Article Snippet: Pharmacological treatment of embryos Dechorionated embryos were incubated in the dark at 25°C in a solution of 0.9% NaCl (under an octane phase) supplemented with DMSO (control), 0.8 mM Dynasore (30 min; Cedarlane) or 0.1 mg/ml leupeptin (3 h; Bioshop).

    Techniques: Staining, Incubation, Expressing, Two Tailed Test

    Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).

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

    Article Title: Functional cleavage of the common cytokine receptor ? chain (?c) by calpain

    doi:

    Figure Lengend Snippet: Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).

    Article Snippet: Cells were lysed with Brij96 lysis buffer containing 5 mM EGTA and 2.5 mM EDTA and a protease inhibitor mix containing 10 μg/ml leupeptin (Boehringer Mannheim), 240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride (ICN), and 10 μg/ml aprotinin (ICN).

    Techniques: In Vitro, Sequencing, Incubation, Protease Inhibitor, Immunoprecipitation, Staining

    In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of leupeptin (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms

    doi: 10.3389/fphar.2018.00819

    Figure Lengend Snippet: In vivo and in vitro effect of tBHQ on autophagic activity. (A) In vivo 2 h blockage of autophagic flux in 6 h starved diabetic mice treated with either vehicle or tBHQ for 3 weeks by intraperitoneal injection of leupeptin (vehicle-group, n = 3; tBHQ, n = 3) or saline (vehicle-group, n = 3; tBHQ, n = 3). Shown are representative blots of MAP1LC3B-II, SQSTM1/p62 and α-tubulin (loading control) and quantification of autophagic flux rate in aortic tissue. (B) Real-time PCR analysis of autophagy genes in VSMC treated with tBHQ 25 μmol/L for 6 h in serum-deprived (-FBS) or serum-supplemented (+FBS) conditions. Data normalized by 18S are expressed as fold increase over respective basal conditions ( n = 9 independent experiments). (C) In vitro blockage of autophagic flux in starved VSMC pretreated with either tBHQ (25 μmol/L, 90 min) or vehicle prior to the addition of lysosomal inhibitors (20 mmol/L NH 4 Cl plus 100 μmol/L leupeptin; N-leup) for 2 h. Shown are representative blots and quantification of autophagic flux rate in VSMC ( n = 7 independent experiments). The rate of autophagic flux is expressed as lysosomal protease inhibitor (leup or N-leup) induced protein accumulation (MAP1LC3B-II or SQSTM1/p62) vs. respective lysosomal protease inhibitor-free condition. Results are expressed as mean ± SEM. ∗ P

    Article Snippet: We injected intraperitoneally a single dose of leupeptin 20 mg/kg b.w. (BP2662, Fisher Scientific, Waltham, MA, United States) or saline into 6 h starved mice during dark cycle, and harvested tissues (aorta and liver) 2 h later.

    Techniques: In Vivo, In Vitro, Activity Assay, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Protease Inhibitor

    Cidea is a short-lived protein and its degradation is proteasome-dependent ( A , B and C ) A 1 μg portion of CMV5–HA–hCidea was co-transfected with 0.5 μg of pEGFP-N1 into HEK-293T, CHO-K1 and H1299 cells in 60-mm-diameter dishes using Dosper reagent. CHX-based protein chase experiments were performed as described below. At 24 h post-transfection, and 1 h prior to the addition of CHX, the medium was replaced with fresh medium, and then CHX was added to a final concentration of 100 μg/ml. Cells were harvested in 0.5 ml of lysis buffer at different time points: 0, 30, 60 and 120 min. Total cell lysates were prepared and analysed by Western blotting [IB (immunoblotting)] using anti-HA and anti-GFP as primary antibodies. The amount of GFP transiently co-expressed by pEGFP-N1 present in total cell lysates was used for the normalization of transfection protein. ( D ) Graph showing band intensities corresponding to HA–Cidea and GFP scanned and quantitified using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. ( E ) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV-HA vector (lane 1) or 1 μg of CMV5–HA–hCidea (lane 2–11) and 0.5 μg of pEGFP-N1 using the calcium phosphate method. After 24 h, CHX was added to 100 μg/ml (lanes 3–11) along with different reagents [DMSO, ethanol, pepstatin (0.5 μg/ml), leupeptin (5 μg/ml), aprotinin (2 μg/ml), ALLN (50 μM), MG132 (10 μM), chloroquine (50 μg/ml) and NH 4 Cl (2.5 mM)] for 2 h. Cells were harvested in 0.5 ml of lysis buffer after the 2 h treatment. Total cell lysates were subjected to Western blotting and detected with anti-HA and anti-GFP antibodies.

    Journal: The Biochemical Journal

    Article Title: Regulation of Cidea protein stability by the ubiquitin-mediated proteasomal degradation pathway

    doi: 10.1042/BJ20070690

    Figure Lengend Snippet: Cidea is a short-lived protein and its degradation is proteasome-dependent ( A , B and C ) A 1 μg portion of CMV5–HA–hCidea was co-transfected with 0.5 μg of pEGFP-N1 into HEK-293T, CHO-K1 and H1299 cells in 60-mm-diameter dishes using Dosper reagent. CHX-based protein chase experiments were performed as described below. At 24 h post-transfection, and 1 h prior to the addition of CHX, the medium was replaced with fresh medium, and then CHX was added to a final concentration of 100 μg/ml. Cells were harvested in 0.5 ml of lysis buffer at different time points: 0, 30, 60 and 120 min. Total cell lysates were prepared and analysed by Western blotting [IB (immunoblotting)] using anti-HA and anti-GFP as primary antibodies. The amount of GFP transiently co-expressed by pEGFP-N1 present in total cell lysates was used for the normalization of transfection protein. ( D ) Graph showing band intensities corresponding to HA–Cidea and GFP scanned and quantitified using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. ( E ) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV-HA vector (lane 1) or 1 μg of CMV5–HA–hCidea (lane 2–11) and 0.5 μg of pEGFP-N1 using the calcium phosphate method. After 24 h, CHX was added to 100 μg/ml (lanes 3–11) along with different reagents [DMSO, ethanol, pepstatin (0.5 μg/ml), leupeptin (5 μg/ml), aprotinin (2 μg/ml), ALLN (50 μM), MG132 (10 μM), chloroquine (50 μg/ml) and NH 4 Cl (2.5 mM)] for 2 h. Cells were harvested in 0.5 ml of lysis buffer after the 2 h treatment. Total cell lysates were subjected to Western blotting and detected with anti-HA and anti-GFP antibodies.

    Article Snippet: Leupeptin and aprotinin were purchased from Amersham.

    Techniques: Transfection, Concentration Assay, Lysis, Western Blot, Software, Plasmid Preparation

    Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM leupeptin with or without 100 nM epoxomycin, according to the same protocol as in A.

    Journal: Molecular Biology of the Cell

    Article Title: Minus end–directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions

    doi: 10.1091/mbc.E14-07-1245

    Figure Lengend Snippet: Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH 4 Cl and 21 μM leupeptin with or without 100 nM epoxomycin, according to the same protocol as in A.

    Article Snippet: Leupeptin was purchased from Peptide Institute (Osaka, Japan).

    Techniques: Transfection, Western Blot

    Propranolol (PPL) modulates ß-AR degradation in human dermal fibroblasts Twenty-four hours after being plated, NF, HSF, and NSF fibroblasts were pretreated for 30 minutes with PPL or vehicle and then treated for the indicated time with a proteasome inhibitor (MG132) (10 μM) or lysosomal inhibitor (leupeptin) (50 μM). Western blotting was performed to determine the expression of ß1-, ß2-, and ß3-ARs in the presence of a. vehicle or b. PPL (10 μM). c. Quantification of ß1-AR band intensity. d. Quantification of ß2-AR band intensity. e. Quantification of ß3-AR band intensity. Data were normalized to control and were presented as mean ± SEM of at least 3 independent experiments. *p

    Journal: The Journal of investigative dermatology

    Article Title: β-Adrenergic receptor trafficking, degradation, and cell-surface expression are altered in dermal fibroblasts from hypertrophic scars

    doi: 10.1016/j.jid.2018.01.037

    Figure Lengend Snippet: Propranolol (PPL) modulates ß-AR degradation in human dermal fibroblasts Twenty-four hours after being plated, NF, HSF, and NSF fibroblasts were pretreated for 30 minutes with PPL or vehicle and then treated for the indicated time with a proteasome inhibitor (MG132) (10 μM) or lysosomal inhibitor (leupeptin) (50 μM). Western blotting was performed to determine the expression of ß1-, ß2-, and ß3-ARs in the presence of a. vehicle or b. PPL (10 μM). c. Quantification of ß1-AR band intensity. d. Quantification of ß2-AR band intensity. e. Quantification of ß3-AR band intensity. Data were normalized to control and were presented as mean ± SEM of at least 3 independent experiments. *p

    Article Snippet: Dermal fibroblasts were treated with MG132 (Calbiochem, Millipore, MA, USA) or Leupeptin (Peptides International, Louisville, KY, USA) ( , ).

    Techniques: Western Blot, Expressing

    Effect of burn trauma on catecholamine-stimulated trafficking and degradation of β2-AR in human dermal fibroblasts Dermal fibroblasts were plated on glass coverslips one day before transfection with the 10 μg/ml ß2-AR–GFP construct or control vector using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). a. Immunofluorescent analysis of NF, HSF, and NSF fibroblasts transfected with ß2-AR–GFP (green) and stimulated for 6 hours with isoproterenol (ISO) or ISO with propranolol (PPL). Lysosomal compartments were visualized with the anti-LAMP2A (red). The bottom two rows show the effect of proteasome (MG132) and lysosome inhibition (Leupeptin-Leup) on ISO-stimulated changes in ß2-AR–GFP trafficking. Arrows point to the areas of colocalization of ß2-AR-GFP to the lysosomes. These experiments have been repeated at least 3 times with 15-20 cells imaged in each preparation. Scale bar, 10 μm. b. Quantification of colocalization (Icorr), presented as the mean ± SEM of 9 separate cells. *p

    Journal: The Journal of investigative dermatology

    Article Title: β-Adrenergic receptor trafficking, degradation, and cell-surface expression are altered in dermal fibroblasts from hypertrophic scars

    doi: 10.1016/j.jid.2018.01.037

    Figure Lengend Snippet: Effect of burn trauma on catecholamine-stimulated trafficking and degradation of β2-AR in human dermal fibroblasts Dermal fibroblasts were plated on glass coverslips one day before transfection with the 10 μg/ml ß2-AR–GFP construct or control vector using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). a. Immunofluorescent analysis of NF, HSF, and NSF fibroblasts transfected with ß2-AR–GFP (green) and stimulated for 6 hours with isoproterenol (ISO) or ISO with propranolol (PPL). Lysosomal compartments were visualized with the anti-LAMP2A (red). The bottom two rows show the effect of proteasome (MG132) and lysosome inhibition (Leupeptin-Leup) on ISO-stimulated changes in ß2-AR–GFP trafficking. Arrows point to the areas of colocalization of ß2-AR-GFP to the lysosomes. These experiments have been repeated at least 3 times with 15-20 cells imaged in each preparation. Scale bar, 10 μm. b. Quantification of colocalization (Icorr), presented as the mean ± SEM of 9 separate cells. *p

    Article Snippet: Dermal fibroblasts were treated with MG132 (Calbiochem, Millipore, MA, USA) or Leupeptin (Peptides International, Louisville, KY, USA) ( , ).

    Techniques: Transfection, Construct, Plasmid Preparation, Inhibition

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

    Journal: The Journal of Biological Chemistry

    Article Title: A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

    doi: 10.1074/jbc.M110.155234

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

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

    Techniques: Activity Assay

    Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with leupeptin (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P

    Journal: Nature immunology

    Article Title: Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells

    doi: 10.1038/s41590-017-0002-1

    Figure Lengend Snippet: Atad3a deficiency induces mitophagy of both healthy mitochondria and damaged mitochondria a , Flow cytometry to distinguish populations with high membrane potential (healthy mitochondria) from those with low potential (damaged mitochondria) among hematopoietic cell populations (above plots) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (left margin). b,c , Mass of healthy mitochondria ( b ) and damaged mitochondria ( c ) in hematopoietic cell populations (horizontal axis) from Atad3a fl/fl or Atad3a fl/fl Mx1Cre mice (key), presented relative to results obtained for Atad3a fl/fl mice. d , Confocal microscopy of the co-localization Tom20 and Lamp2a in HEK293T cells expressing (left margin) control (non-targeting) shRNA (shControl) or Atad3a -specific shRNA (shAtad3a) and treated with leupeptin (bottom two rows) or not (top two rows). Scale bar, 20 μm. e , Frequency of co-localized Tom20 and Lamp2a in cells as in d . * P

    Article Snippet: For immunofluorescence staining, HEK293T cells in which Atad3a was knocked down or not were seeded in the culture chamber and were treated with or without 10 μM leupeptin for 3 h, then fixed with 4% PFA, permeabilized with 0.1% Triton and stained with anti-Tom20 (sc-17764, Santa Cruz) and anti-Lamp2a (ab18528, Abcam).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Confocal Microscopy, Expressing, shRNA

    Inhibitory analysis of L1 proteolysis. HeLa (A) or HaCaT (B) cells were infected with HPV16 PsV in the presence or absence of the following inhibitors: NH 4 Cl (20 mM), bafilomycin A1 (10 nM), E-64 (100 μM), leupeptin (100 μM), CA-074 or

    Journal: Journal of Virology

    Article Title: Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

    doi: 10.1128/JVI.00234-15

    Figure Lengend Snippet: Inhibitory analysis of L1 proteolysis. HeLa (A) or HaCaT (B) cells were infected with HPV16 PsV in the presence or absence of the following inhibitors: NH 4 Cl (20 mM), bafilomycin A1 (10 nM), E-64 (100 μM), leupeptin (100 μM), CA-074 or

    Article Snippet: AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride], leupeptin, ZnCl2 , and ZnSO4 were from Applichem.

    Techniques: Infection

    Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor (leupeptin), 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p

    Journal: PLoS Pathogens

    Article Title: Post-Translational Control of IL-1? via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53

    doi: 10.1371/journal.ppat.1003536

    Figure Lengend Snippet: Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor (leupeptin), 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p

    Article Snippet: Inhibitors of autophagy used: 1 mM 3-methyladenine (Enzo Life Sciences) incubated for 8 h and 100 nM bafilomycin (Enzo Life Sciences) incubated for 8 h. Inhibitors of lysosome/protease activity: 20 µM of Calpain inhibitor PD 150,606 (Adipogen), 25 µM of cathepsin B inhibitor CA-074 (Enzo Life Sciences), 10 µM of Leupeptin (Biomol), 20 µM Vincristine (Enzo Life Sciences).

    Techniques: Confocal Microscopy, Immunostaining, Transduction, Expressing, Blocking Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activity Assay, Fluorescence, Incubation, Staining, Protease Inhibitor, High Throughput Screening Assay, Microscopy, Imaging

    Abrogation of pro-HGF mediated c-met activation and breast cancer cell proliferation using matriptase inhibitors (a) Matriptase sufficient mouse mammary carcinoma cells were isolated from Mat-PymT + mice and exposed to pro-HGF or pre-cleaved active HGF with and without matriptase inhibitors added concomitantly (60 nM HAI-1, 40 nM HAI-2 or 20 uM leupeptin). Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (b) Mouse mammary carcinoma cells or human breast cancer BT20 cells were serum starved and pre-incubated with 1 μM IN-1 synthetic matriptase inhibitor for 30 min before exposure to active HGF, pro-HGF, or no growth factors. Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (c) BT20 cells were serum starved and exposed to either active HGF (gray bars), pro-HGF (white bars), or no growth factor (black bars) for 24 h and counted. The synthetic selective matriptase inhibitor IN-1 was added at 10 nM, 100 nM, 1 μM, or 10 μM concomitantly with pro-HGF/HGF. In the mock control vehicle (PBS) was added. Graphs show the increase in cell numbers at 24 h after stimulation (triplicates) (*P

    Journal: Nature communications

    Article Title: Targeting matriptase in breast cancer abrogates tumor progression via impairment of stromal-epithelial growth factor signaling

    doi: 10.1038/ncomms7776

    Figure Lengend Snippet: Abrogation of pro-HGF mediated c-met activation and breast cancer cell proliferation using matriptase inhibitors (a) Matriptase sufficient mouse mammary carcinoma cells were isolated from Mat-PymT + mice and exposed to pro-HGF or pre-cleaved active HGF with and without matriptase inhibitors added concomitantly (60 nM HAI-1, 40 nM HAI-2 or 20 uM leupeptin). Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (b) Mouse mammary carcinoma cells or human breast cancer BT20 cells were serum starved and pre-incubated with 1 μM IN-1 synthetic matriptase inhibitor for 30 min before exposure to active HGF, pro-HGF, or no growth factors. Cells were lysed at the indicated time points after growth factor stimulation and analyzed for total and activated c-Met by western blotting. (c) BT20 cells were serum starved and exposed to either active HGF (gray bars), pro-HGF (white bars), or no growth factor (black bars) for 24 h and counted. The synthetic selective matriptase inhibitor IN-1 was added at 10 nM, 100 nM, 1 μM, or 10 μM concomitantly with pro-HGF/HGF. In the mock control vehicle (PBS) was added. Graphs show the increase in cell numbers at 24 h after stimulation (triplicates) (*P

    Article Snippet: Cells were then serum starved for a minimum of 3 h. The media was changed to either fresh serum free media, serum free media with 100 ng/ml recombinant active two-chain HGF/SF (R & D Systems, Minneapolis, MN) or pro-HGF/SF with or without either 2 μM Aprotinin (MP Biomedicals, LLC, Solon, OH), 20 μM Leupeptin (Fisher Bioreagents, Fair Lawn, New Jersey), 60 nM HAI-1, 40 nM HAI-2 (R & D Systems), or 1 μM IN-1 .

    Techniques: Activation Assay, Isolation, Mouse Assay, Western Blot, Incubation

    Evaluation of the overall performance of the nanofractionation system using the plasmin inhibitor leupeptin, which was nanofractionated at five concentrations injected. ( a ) 6.3 μM. ( b ) 25 μM. ( c ) 50 μM. ( d ) 100 μM. ( e ) 200 μM. ( f ) The extracted ion chromatogram ( m/z 427.3, [M + H] + ) of the analysis of 100 μM leupeptin with online LC-MS. RFU, relative fluorescence units

    Journal: Analytical and Bioanalytical Chemistry

    Article Title: Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms

    doi: 10.1007/s00216-018-1253-x

    Figure Lengend Snippet: Evaluation of the overall performance of the nanofractionation system using the plasmin inhibitor leupeptin, which was nanofractionated at five concentrations injected. ( a ) 6.3 μM. ( b ) 25 μM. ( c ) 50 μM. ( d ) 100 μM. ( e ) 200 μM. ( f ) The extracted ion chromatogram ( m/z 427.3, [M + H] + ) of the analysis of 100 μM leupeptin with online LC-MS. RFU, relative fluorescence units

    Article Snippet: Leupeptin hydrochloride (dissolved in Milli-Q water to obtain a stock of 10 mM), 1,10-phenanthroline, bovine serum albumin (BSA), calcium chloride, DMSO, EDTA, iodoacetamide, trypsin from bovine pancreas, and Trizma were all obtained from Sigma (Zwijndrecht, The Netherlands).

    Techniques: Injection, Liquid Chromatography with Mass Spectroscopy, Fluorescence

    Assessment of the bioassay performance. The Z ′-factor for the bioassay was calculated to be 0.74. Positive control ( C+ ) wells contain mobile phase A ( n = 96); negative control ( C− ) wells contain 200 μM leupeptin ( n = 96). The wells were evaporated to dryness prior to the bioassay

    Journal: Analytical and Bioanalytical Chemistry

    Article Title: Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms

    doi: 10.1007/s00216-018-1253-x

    Figure Lengend Snippet: Assessment of the bioassay performance. The Z ′-factor for the bioassay was calculated to be 0.74. Positive control ( C+ ) wells contain mobile phase A ( n = 96); negative control ( C− ) wells contain 200 μM leupeptin ( n = 96). The wells were evaporated to dryness prior to the bioassay

    Article Snippet: Leupeptin hydrochloride (dissolved in Milli-Q water to obtain a stock of 10 mM), 1,10-phenanthroline, bovine serum albumin (BSA), calcium chloride, DMSO, EDTA, iodoacetamide, trypsin from bovine pancreas, and Trizma were all obtained from Sigma (Zwijndrecht, The Netherlands).

    Techniques: Positive Control, Negative Control