leupeptin  (Millipore)


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    Leupeptin
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    Millipore leupeptin
    Leupeptin

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    Average 99 stars, based on 1366 article reviews
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    leupeptin - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "MEK Inhibition Leads To Lysosome-Mediated Na+/I- Symporter Protein Degradation In Human Breast Cancer Cells"

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

    Journal: Endocrine-related cancer

    doi: 10.1530/ERC-12-0342

    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).
    Figure Legend 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).

    Techniques Used: Inhibition, Western Blot, Cell Culture

    2) Product Images from "USP15-dependent lysosomal pathway controls p53-R175H turnover in ovarian cancer cells"

    Article Title: USP15-dependent lysosomal pathway controls p53-R175H turnover in ovarian cancer cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03599-w

    MCB-613 causes lysosome-mediated degradation on p53-R175H. a Effect of MCB-613 (2 h) on p53 mRNA levels in ALST and TYK-Nu cells. Values are normalized mean ± s.e.m. ( n = 3). b – d siRNA-mediated knockdown (48 h) of SRC-1 ( b ), SRC-2 ( c ), and SRC-3 ( d ) had no effect on p53-WT (ALST cells) and p53-R175H (TYK-Nu cells) levels. e shRNA-mediated knockdown of SRC3 (72 h) in TYK-Nu cells does not affect p53-R175H levels. f SRC-3 inhibitor SI2 (50 nM, 2 h) has no effect on p53-R175H levels in TYK-Nu cells. g , h MCB-613 caused a g decrease in the half-life of p53-R175H and h increase in the half-life of p53-WT. i Proteasome inhibitor MG132 does not inhibit MCB-613 induced turnover of p53-R175H. j Lysosome inhibitors (pepstatin A, Leupeptin, and E-64D) rescued MCB-613 induced turnover of p53-R175H
    Figure Legend Snippet: MCB-613 causes lysosome-mediated degradation on p53-R175H. a Effect of MCB-613 (2 h) on p53 mRNA levels in ALST and TYK-Nu cells. Values are normalized mean ± s.e.m. ( n = 3). b – d siRNA-mediated knockdown (48 h) of SRC-1 ( b ), SRC-2 ( c ), and SRC-3 ( d ) had no effect on p53-WT (ALST cells) and p53-R175H (TYK-Nu cells) levels. e shRNA-mediated knockdown of SRC3 (72 h) in TYK-Nu cells does not affect p53-R175H levels. f SRC-3 inhibitor SI2 (50 nM, 2 h) has no effect on p53-R175H levels in TYK-Nu cells. g , h MCB-613 caused a g decrease in the half-life of p53-R175H and h increase in the half-life of p53-WT. i Proteasome inhibitor MG132 does not inhibit MCB-613 induced turnover of p53-R175H. j Lysosome inhibitors (pepstatin A, Leupeptin, and E-64D) rescued MCB-613 induced turnover of p53-R175H

    Techniques Used: shRNA

    3) Product Images from "Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis"

    Article Title: Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201404223

    Lysosomal localization of LAMP2A in Ctns − / − cells is rescued by inhibition of lysosomal proteases WT, Ctns −/− or Ctns −/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns − / − cells. Scale bars: 5 μm. Inset scale bars: 2 μm.
    Figure Legend Snippet: Lysosomal localization of LAMP2A in Ctns − / − cells is rescued by inhibition of lysosomal proteases WT, Ctns −/− or Ctns −/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns − / − cells. Scale bars: 5 μm. Inset scale bars: 2 μm.

    Techniques Used: Inhibition, Confocal Microscopy

    4) Product Images from "Endocytosis of Secreted Carboxyl Ester Lipase in a Syndrome of Diabetes and Pancreatic Exocrine Dysfunction"

    Article Title: Endocytosis of Secreted Carboxyl Ester Lipase in a Syndrome of Diabetes and Pancreatic Exocrine Dysfunction

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.574244

    Co-localization of CEL-MUT- and CEL-TRUNC-positive vacuoles with LAMP1. A , stably transfected HEK293 cells were treated with vehicle (DMSO = untreated), 10 μ m MG132, or 100 μg/ml leupeptin for 3 h. The cells were fixed and stained for
    Figure Legend Snippet: Co-localization of CEL-MUT- and CEL-TRUNC-positive vacuoles with LAMP1. A , stably transfected HEK293 cells were treated with vehicle (DMSO = untreated), 10 μ m MG132, or 100 μg/ml leupeptin for 3 h. The cells were fixed and stained for

    Techniques Used: Stable Transfection, Transfection, Staining

    CEL-MUT endocytosed by HeLa cells accumulates in LAMP1-positive vacuoles. HeLa cells were cultured in conditioned medium from HEK293 cells stably expressing CEL-MUT for 3 h in the presence of vehicle (DMSO), 100 μg/ml leupeptin, 100 n m BafA1,
    Figure Legend Snippet: CEL-MUT endocytosed by HeLa cells accumulates in LAMP1-positive vacuoles. HeLa cells were cultured in conditioned medium from HEK293 cells stably expressing CEL-MUT for 3 h in the presence of vehicle (DMSO), 100 μg/ml leupeptin, 100 n m BafA1,

    Techniques Used: Cell Culture, Stable Transfection, Expressing

    5) Product Images from "Proteolysis of the Ebola Virus Glycoproteins Enhances Virus Binding and Infectivity ▿"

    Article Title: Proteolysis of the Ebola Virus Glycoproteins Enhances Virus Binding and Infectivity ▿

    Journal:

    doi: 10.1128/JVI.01170-07

    Proteolysis of Ebola virus GP and inhibition of infection using cathepsin inhibitors. (A) VeroE6 cells were treated with NH 4 Cl (50 mM), leupeptin (10 μM), E64-d (10 μM), Z-LLL-FMK (10 μM), or CA074 (100 μM) for 1 h at 37°C
    Figure Legend Snippet: Proteolysis of Ebola virus GP and inhibition of infection using cathepsin inhibitors. (A) VeroE6 cells were treated with NH 4 Cl (50 mM), leupeptin (10 μM), E64-d (10 μM), Z-LLL-FMK (10 μM), or CA074 (100 μM) for 1 h at 37°C

    Techniques Used: Inhibition, Infection

    6) Product Images from "AXL Mediates Esophageal Adenocarcinoma Cell Invasion through Regulation of Extracellular Acidification and Lysosome Trafficking"

    Article Title: AXL Mediates Esophageal Adenocarcinoma Cell Invasion through Regulation of Extracellular Acidification and Lysosome Trafficking

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.08.005

    Pharmacological inhibition of cathepsin B activity impairs AXL-dependent cell invasion in EAC cells. (A-F) Transwell invasion assay in the presence of leupeptin. (A) Transwell invasion assay representative images (20×) of SK-GT-4 cells or (D) FLO-1 cells treated with vehicle or leupeptin (20 μM) for 48 hours. (B) Transwell invasion assay quantification as the number of invading SK-GT-4 cells or (F) FLO-1 cells per microscopic field after treatment with vehicle or leupeptin. Data are presented as median ± SD. (C) Trypan blue viability assay of SK-GT-4 cells or (F) FLO-1 cells following treatment with vehicle or leupeptin. (G-L) Transwell invasion assay in the presence of CA-074. (G) Transwell invasion assay representative images (20×) of SK-GT-4 cells or (J) FLO-1 cells treated with vehicle or CA-074 (10 μM) for 48 hours. (H) Transwell invasion assay quantification as the number of invading SK-GT-4 cells or (K) FLO-1 cells per microscopic field after treatment with vehicle or CA-074. Data are presented as median ± SD. (I) Trypan blue-viability assay of SK-GT-4 cells or (L) FLO-1 cells following treatment with vehicle or CA-074.
    Figure Legend Snippet: Pharmacological inhibition of cathepsin B activity impairs AXL-dependent cell invasion in EAC cells. (A-F) Transwell invasion assay in the presence of leupeptin. (A) Transwell invasion assay representative images (20×) of SK-GT-4 cells or (D) FLO-1 cells treated with vehicle or leupeptin (20 μM) for 48 hours. (B) Transwell invasion assay quantification as the number of invading SK-GT-4 cells or (F) FLO-1 cells per microscopic field after treatment with vehicle or leupeptin. Data are presented as median ± SD. (C) Trypan blue viability assay of SK-GT-4 cells or (F) FLO-1 cells following treatment with vehicle or leupeptin. (G-L) Transwell invasion assay in the presence of CA-074. (G) Transwell invasion assay representative images (20×) of SK-GT-4 cells or (J) FLO-1 cells treated with vehicle or CA-074 (10 μM) for 48 hours. (H) Transwell invasion assay quantification as the number of invading SK-GT-4 cells or (K) FLO-1 cells per microscopic field after treatment with vehicle or CA-074. Data are presented as median ± SD. (I) Trypan blue-viability assay of SK-GT-4 cells or (L) FLO-1 cells following treatment with vehicle or CA-074.

    Techniques Used: Inhibition, Activity Assay, Transwell Invasion Assay, Viability Assay

    7) Product Images from "Activation of pro-survival metabolic networks by 1,25(OH)2D3 does not hamper the sensitivity of breast cancer cells to chemotherapeutics"

    Article Title: Activation of pro-survival metabolic networks by 1,25(OH)2D3 does not hamper the sensitivity of breast cancer cells to chemotherapeutics

    Journal: Cancer & Metabolism

    doi: 10.1186/s40170-018-0183-6

    1,25(OH) 2 D 3 induces proteasomal degradation of TXNIP in MCF-7 cells. a The reduction in TXNIP protein levels by 1,25(OH) 2 D 3 (100 nM) in MCF-7 cells is rescued by MG-132 (5 μM) or 2-deoxyglucose (10 mM), but not leupeptin (20 μM). The various molecules were added to the conditioned medium of DMSO- and 1,25(OH) 2 D 3 -treated (66 h) MCF-7 cells, for an additional 6 h. b , c ITCH mRNA and protein expression is not markedly influenced by 1,25(OH) 2 D 3 . Relative expression was calculated using the ∆∆Ct method with vinculin as the housekeeping gene. Error bars ± SD; n > 3. d Overall protein ubiquitination in MCF-7 cells was not changed by 1,25(OH) 2 D 3 treatment. e Co-immunoprecipitation studies illustrate that the TXNIP-ITCH interaction is not altered by 1,25(OH) 2 D 3 treatment of MCF-7 cells. f Negative regulation of TXNIP protein expression by 1,25(OH) 2 D 3 is observed in MCF-7 cells with knocked-down AMPKα1 levels. g The non-calcemic 1,25(OH) 2 D 3 analogue, calcipotriol (100 nM; 72 h) induces similar effects on TXNIP expression as 1,25(OH) 2 D 3 . The cell permeable Ca 2+ chelator BAPTA-AM (20 μM) does not hamper 1 ,25(OH) 2 D 3 ’s effects on TXNIP expression. BAPTA-AM was added to the conditioned medium of DMSO- and 1,25(OH) 2 D 3 -treated MCF-7 cells, 2 h prior to the end of the treatment period (72 h)
    Figure Legend Snippet: 1,25(OH) 2 D 3 induces proteasomal degradation of TXNIP in MCF-7 cells. a The reduction in TXNIP protein levels by 1,25(OH) 2 D 3 (100 nM) in MCF-7 cells is rescued by MG-132 (5 μM) or 2-deoxyglucose (10 mM), but not leupeptin (20 μM). The various molecules were added to the conditioned medium of DMSO- and 1,25(OH) 2 D 3 -treated (66 h) MCF-7 cells, for an additional 6 h. b , c ITCH mRNA and protein expression is not markedly influenced by 1,25(OH) 2 D 3 . Relative expression was calculated using the ∆∆Ct method with vinculin as the housekeeping gene. Error bars ± SD; n > 3. d Overall protein ubiquitination in MCF-7 cells was not changed by 1,25(OH) 2 D 3 treatment. e Co-immunoprecipitation studies illustrate that the TXNIP-ITCH interaction is not altered by 1,25(OH) 2 D 3 treatment of MCF-7 cells. f Negative regulation of TXNIP protein expression by 1,25(OH) 2 D 3 is observed in MCF-7 cells with knocked-down AMPKα1 levels. g The non-calcemic 1,25(OH) 2 D 3 analogue, calcipotriol (100 nM; 72 h) induces similar effects on TXNIP expression as 1,25(OH) 2 D 3 . The cell permeable Ca 2+ chelator BAPTA-AM (20 μM) does not hamper 1 ,25(OH) 2 D 3 ’s effects on TXNIP expression. BAPTA-AM was added to the conditioned medium of DMSO- and 1,25(OH) 2 D 3 -treated MCF-7 cells, 2 h prior to the end of the treatment period (72 h)

    Techniques Used: Expressing, Immunoprecipitation

    8) Product Images from "Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins"

    Article Title: Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051287

    Efficient presentation of virion-derived antigens after receptor-mediated uptake. (A) MiniLCL JM were incubated with the anti-CD21 antibody FE8 or an isotype control (Iso1) antibody and pulsed with virus supernatant (EBV). In parallel, virus supernatant was incubated with the anti-BLLF1 antibody 72A1 or an isotype control antibody (Iso2) and pulsed onto miniLCL. After 24 h of incubation, virus-pulsed miniLCL were probed with the T cells. (B) MiniLCL, PBMCs, and dendritic cells (DC) were incubated with increasing amounts of purified virus for 24 h and probed with BLLF1-1H2 T cells. (C) The three types of APCs as in B were incubated with increasing amounts of purified BLLF1 mutant protein lacking the CD21 binding domain for 24 h and subsequently probed with the BLLF1-1H2 T cells. (D) PBMCs were separated into CD19 + and CD19 − cell fractions by magnetic sorting and subsequently incubated for 24 h with increasing amounts of purified viral particles before addition of glycoprotein-specific T cells. (E) MiniLCL were pulsed for 24 h with increasing amounts of EBV geq and probed with BLLF1- and BALF4-specific T cells as indicated. (F) MiniLCL JM were incubated for 24 h with purified viral supernatant in the absence or presence of leupeptin or chloroquine. After the incubation period, unbound virus and inhibitors were removed by washing, and the cells were fixed and probed with the glycoprotein-specific T cells.
    Figure Legend Snippet: Efficient presentation of virion-derived antigens after receptor-mediated uptake. (A) MiniLCL JM were incubated with the anti-CD21 antibody FE8 or an isotype control (Iso1) antibody and pulsed with virus supernatant (EBV). In parallel, virus supernatant was incubated with the anti-BLLF1 antibody 72A1 or an isotype control antibody (Iso2) and pulsed onto miniLCL. After 24 h of incubation, virus-pulsed miniLCL were probed with the T cells. (B) MiniLCL, PBMCs, and dendritic cells (DC) were incubated with increasing amounts of purified virus for 24 h and probed with BLLF1-1H2 T cells. (C) The three types of APCs as in B were incubated with increasing amounts of purified BLLF1 mutant protein lacking the CD21 binding domain for 24 h and subsequently probed with the BLLF1-1H2 T cells. (D) PBMCs were separated into CD19 + and CD19 − cell fractions by magnetic sorting and subsequently incubated for 24 h with increasing amounts of purified viral particles before addition of glycoprotein-specific T cells. (E) MiniLCL were pulsed for 24 h with increasing amounts of EBV geq and probed with BLLF1- and BALF4-specific T cells as indicated. (F) MiniLCL JM were incubated for 24 h with purified viral supernatant in the absence or presence of leupeptin or chloroquine. After the incubation period, unbound virus and inhibitors were removed by washing, and the cells were fixed and probed with the glycoprotein-specific T cells.

    Techniques Used: Derivative Assay, Incubation, Purification, Mutagenesis, Binding Assay

    9) Product Images from "Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability"

    Article Title: Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.153

    Palmitoylation protects the Fas receptor from lysosomal degradation. ( a ) The lysosomal degradation pathway inhibitors Leupeptin (100 and 300 μ M), chloroquine (50 and 100 μ M) and bafilomycin A1 (30, 100 and 300 nM)
    Figure Legend Snippet: Palmitoylation protects the Fas receptor from lysosomal degradation. ( a ) The lysosomal degradation pathway inhibitors Leupeptin (100 and 300 μ M), chloroquine (50 and 100 μ M) and bafilomycin A1 (30, 100 and 300 nM)

    Techniques Used:

    10) Product Images from "Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells"

    Article Title: Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200105138

    Wortmannin induces formation of distinct vacuole populations, but does not alter mature lysosome morphology. In panels a–d, cells were treated for 90 min with 100 nM wortmannin. The relative distributions of endolyn-78 to ASGP-R (a and b) or M6P-R (c and d) are shown. Arrows in a and b are pointing to structures that only contain ASGP-R, and in c and d that contain only M6P-R. In e–k, cells were treated for 180 min with 100 nM wortmannin. In e and f, 50 nM lysotracker was added in the final 30 min of wortmannin treatment. Lysotracker staining of acidic compartments in live cells is shown in panel f, and the corresponding phase image in panel e. Enlarged images of wortmannin-treated cells double- labeled for endolyn-78 (h) and cathepsin D (cath D, g) are shown. Arrowheads indicate small puncta containing endolyn at the surface and cathepsin D in the lumen. In panels i–k, 50 μM leupeptin was included hourly in the wortmannin incubations to inhibit acid hydrolase activity. Arrows are pointing to small puncta that contain 5′NT in panel i. Arrows in the enlarged, double-labeled images in j and k are pointing to the small 5′NT-positive puncta that also contain cathepsin D. The data are representative of at least three experiments. Bars, (a–f and i) 10 μm; (g and h) 2 μm; and (j and k) 5 μm.
    Figure Legend Snippet: Wortmannin induces formation of distinct vacuole populations, but does not alter mature lysosome morphology. In panels a–d, cells were treated for 90 min with 100 nM wortmannin. The relative distributions of endolyn-78 to ASGP-R (a and b) or M6P-R (c and d) are shown. Arrows in a and b are pointing to structures that only contain ASGP-R, and in c and d that contain only M6P-R. In e–k, cells were treated for 180 min with 100 nM wortmannin. In e and f, 50 nM lysotracker was added in the final 30 min of wortmannin treatment. Lysotracker staining of acidic compartments in live cells is shown in panel f, and the corresponding phase image in panel e. Enlarged images of wortmannin-treated cells double- labeled for endolyn-78 (h) and cathepsin D (cath D, g) are shown. Arrowheads indicate small puncta containing endolyn at the surface and cathepsin D in the lumen. In panels i–k, 50 μM leupeptin was included hourly in the wortmannin incubations to inhibit acid hydrolase activity. Arrows are pointing to small puncta that contain 5′NT in panel i. Arrows in the enlarged, double-labeled images in j and k are pointing to the small 5′NT-positive puncta that also contain cathepsin D. The data are representative of at least three experiments. Bars, (a–f and i) 10 μm; (g and h) 2 μm; and (j and k) 5 μm.

    Techniques Used: Staining, Labeling, Activity Assay

    11) Product Images from "Mannose 6-Phosphate/Insulin-like Growth Factor-II Receptor Targets the Urokinase Receptor to Lysosomes via a Novel Binding Interaction "

    Article Title: Mannose 6-Phosphate/Insulin-like Growth Factor-II Receptor Targets the Urokinase Receptor to Lysosomes via a Novel Binding Interaction

    Journal: The Journal of Cell Biology

    doi:

    Colocalization of uPAR and LAMP-2 in leupeptin and pepstatin A–treated clone Cc2 cells. uPAR, 5-nm gold particles, LAMP-2, 10-nm gold particles. ( A ) Intense labeling of uPAR in the matrix of electron dense cytoplasmic vacuoles. ( B ) Colocalization of uPAR ( arrowheads ) and the lysosomal marker LAMP-2 ( arrows ). Bar, 0.25 μm.
    Figure Legend Snippet: Colocalization of uPAR and LAMP-2 in leupeptin and pepstatin A–treated clone Cc2 cells. uPAR, 5-nm gold particles, LAMP-2, 10-nm gold particles. ( A ) Intense labeling of uPAR in the matrix of electron dense cytoplasmic vacuoles. ( B ) Colocalization of uPAR ( arrowheads ) and the lysosomal marker LAMP-2 ( arrows ). Bar, 0.25 μm.

    Techniques Used: Labeling, Marker

    Labeling of uPAR in cultured fibroblasts. HT1080 fibroblasts were cultured in the presence of 50 μg leupeptin and 66 μg/ml pepstatin A for 17 h ( left panels ), or with 400 nM RAP for 12 h, followed by leupeptin and pepstatin A for 17 h in the continuous presence of RAP ( right panels ). The upper panels show uPAR labeling using the monoclonal anti-uPAR antibody R2 and rhodamine coupled goat anti–mouse IgG. The middle panels show LAMP-1 labeling using specific rabbit anti– LAMP-1 serum and FITC-labeled swine anti–rabbit IgG. The lower panels show a double exposure of the uPAR and LAMP-1 staining with colocalization appearing in yellow.
    Figure Legend Snippet: Labeling of uPAR in cultured fibroblasts. HT1080 fibroblasts were cultured in the presence of 50 μg leupeptin and 66 μg/ml pepstatin A for 17 h ( left panels ), or with 400 nM RAP for 12 h, followed by leupeptin and pepstatin A for 17 h in the continuous presence of RAP ( right panels ). The upper panels show uPAR labeling using the monoclonal anti-uPAR antibody R2 and rhodamine coupled goat anti–mouse IgG. The middle panels show LAMP-1 labeling using specific rabbit anti– LAMP-1 serum and FITC-labeled swine anti–rabbit IgG. The lower panels show a double exposure of the uPAR and LAMP-1 staining with colocalization appearing in yellow.

    Techniques Used: Labeling, Cell Culture, Staining

    Colocalization of uPAR and CIMPR in Cc2 cells. uPAR, 5-nm gold particles; CIMPR, 10-nm gold particles. ( A ) uPAR ( arrowheads ) and CIMPR ( arrows ) are seen on the plasma membrane, including microvilli. ( B ) Colocalization of uPAR ( arrowheads ) and CIMPR ( arrows ) in late endosomes. The incubation was performed with leupeptin (50 μg/ml) and pepstatin A (67 μg/ml). Bar, 0.25 μm.
    Figure Legend Snippet: Colocalization of uPAR and CIMPR in Cc2 cells. uPAR, 5-nm gold particles; CIMPR, 10-nm gold particles. ( A ) uPAR ( arrowheads ) and CIMPR ( arrows ) are seen on the plasma membrane, including microvilli. ( B ) Colocalization of uPAR ( arrowheads ) and CIMPR ( arrows ) in late endosomes. The incubation was performed with leupeptin (50 μg/ml) and pepstatin A (67 μg/ml). Bar, 0.25 μm.

    Techniques Used: Incubation

    12) Product Images from "Phosphorylation of Protease-activated Receptor-2 Differentially Regulates Desensitization and Internalization *"

    Article Title: Phosphorylation of Protease-activated Receptor-2 Differentially Regulates Desensitization and Internalization *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.048942

    Co-localization of PAR2 wild-type and 0P mutant with LAMP1. HeLa cells stably expressing FLAG-tagged PAR2 wild-type ( WT ) or 0P mutant were pre-treated with or without 2 m m leupeptin for 1 h at 37 °C. Cells were incubated with polyclonal anti-FLAG
    Figure Legend Snippet: Co-localization of PAR2 wild-type and 0P mutant with LAMP1. HeLa cells stably expressing FLAG-tagged PAR2 wild-type ( WT ) or 0P mutant were pre-treated with or without 2 m m leupeptin for 1 h at 37 °C. Cells were incubated with polyclonal anti-FLAG

    Techniques Used: Mutagenesis, Stable Transfection, Expressing, Incubation

    13) Product Images from "Downregulation of p53 drives autophagy during human trophoblast differentiation"

    Article Title: Downregulation of p53 drives autophagy during human trophoblast differentiation

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-017-2695-6

    p53 and autophagic activity in primary trophoblast culture. Primary term trophoblasts were seeded 1 day prior to time point 0 h. Culture supernatants and cell lysates were collected at indicated time points. a Secretion of βhCG increased over time and suggested spontaneous differentiation of cultured trophoblasts. Immunoblotting ( b ) and band densitometry showed declining p53 levels ( c ), while LC3B-II levels ( d ) increased during culture. Incubation of trophoblasts with leupeptin (100 µM) did not change p53 protein, whereas LC3B-II levels ( e ) increased after 48 h incubation. Nutlin-3a treatment (10 µM, 48 h) increased p53 and decreased LC3B-II levels ( f ) when compared to control. Data in a , c and d are presented as mean ± SEM from three different trophoblast preparations. * p ≤ 0.05
    Figure Legend Snippet: p53 and autophagic activity in primary trophoblast culture. Primary term trophoblasts were seeded 1 day prior to time point 0 h. Culture supernatants and cell lysates were collected at indicated time points. a Secretion of βhCG increased over time and suggested spontaneous differentiation of cultured trophoblasts. Immunoblotting ( b ) and band densitometry showed declining p53 levels ( c ), while LC3B-II levels ( d ) increased during culture. Incubation of trophoblasts with leupeptin (100 µM) did not change p53 protein, whereas LC3B-II levels ( e ) increased after 48 h incubation. Nutlin-3a treatment (10 µM, 48 h) increased p53 and decreased LC3B-II levels ( f ) when compared to control. Data in a , c and d are presented as mean ± SEM from three different trophoblast preparations. * p ≤ 0.05

    Techniques Used: Activity Assay, Cell Culture, Incubation

    Nutlin-3a increased p53 and reduced LC3B-II levels in placental explants. Incubation of human first trimester placental explants with leupeptin (100 µM) and subsequent immunoblotting ( a ) showed unchanged p53 protein ( b ) and increased LC3B-II levels ( c ) after 48 h incubation, suggesting basal autophagic activity in placental explant culture. Nutlin-3a treatment (10 µM, 48 h) substantially increased placental p53 levels, whereas LC3B-II levels at the same time declined when compared to solvent control ( d , e , f ). Immunohistochemistry of control ( g ) and Nutlin-3a treated placental explants ( h ) revealed increased p53 staining intensity of villous cytotrophoblast nuclei (open arrowhead), while nuclei of the syncytiotrophoblast (arrowhead) remained unstained in response to Nutlin-3a. Scale bar in h represents 100 µm. Data in b and c are presented as mean ± SEM from three and those in e and f from four different patients. * p ≤ 0.05
    Figure Legend Snippet: Nutlin-3a increased p53 and reduced LC3B-II levels in placental explants. Incubation of human first trimester placental explants with leupeptin (100 µM) and subsequent immunoblotting ( a ) showed unchanged p53 protein ( b ) and increased LC3B-II levels ( c ) after 48 h incubation, suggesting basal autophagic activity in placental explant culture. Nutlin-3a treatment (10 µM, 48 h) substantially increased placental p53 levels, whereas LC3B-II levels at the same time declined when compared to solvent control ( d , e , f ). Immunohistochemistry of control ( g ) and Nutlin-3a treated placental explants ( h ) revealed increased p53 staining intensity of villous cytotrophoblast nuclei (open arrowhead), while nuclei of the syncytiotrophoblast (arrowhead) remained unstained in response to Nutlin-3a. Scale bar in h represents 100 µm. Data in b and c are presented as mean ± SEM from three and those in e and f from four different patients. * p ≤ 0.05

    Techniques Used: Incubation, Activity Assay, Immunohistochemistry, Staining

    Autophagic activity is increased in BeWo differentiation. Immunoblotting ( a ) and band densitometry ( b ) showed that leupeptin (100 µM) treatment further augmented forskolin-induced (20 µM, 48 h) increase of LC3B-II levels, when compared to forskolin treatment alone. Transfection of BeWo cells with a LC3B-GFP construct showed moderate LC3B puncta in vehicle control ( c , DMSO, 0.1%), which increased after leupeptin (100 µM) treatment ( d ). Forskolin stimulation ( e ) increased LC3B puncta compared to vehicle control, and further increased after both forskolin and leupeptin administration ( f ). Software-based image analysis of LC3B-GFP puncta ( g ) by trend confirmed data obtained with immunoblotting. Scale bar in c represents 50 µm. Data in b are presented as mean ± SEM from three independent experiments, using different cell passages. * p ≤ 0.05
    Figure Legend Snippet: Autophagic activity is increased in BeWo differentiation. Immunoblotting ( a ) and band densitometry ( b ) showed that leupeptin (100 µM) treatment further augmented forskolin-induced (20 µM, 48 h) increase of LC3B-II levels, when compared to forskolin treatment alone. Transfection of BeWo cells with a LC3B-GFP construct showed moderate LC3B puncta in vehicle control ( c , DMSO, 0.1%), which increased after leupeptin (100 µM) treatment ( d ). Forskolin stimulation ( e ) increased LC3B puncta compared to vehicle control, and further increased after both forskolin and leupeptin administration ( f ). Software-based image analysis of LC3B-GFP puncta ( g ) by trend confirmed data obtained with immunoblotting. Scale bar in c represents 50 µm. Data in b are presented as mean ± SEM from three independent experiments, using different cell passages. * p ≤ 0.05

    Techniques Used: Activity Assay, Transfection, Construct, Software

    14) Product Images from "Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process"

    Article Title: Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2012.10

    Ca074Me delays B10-induced cell death and transiently reduces Bid degradation, caspase-3 activation and mitochondrial membrane depolarization. ( A ) U87MG cells were incubated with 18 μ M B10 or DMSO in combination with Ca074Me (10 μ M), E64 (50 μ M) or leupeptin (100 μ M). Cell viability was quantified by crystal violet staining after 24 h (black bars) and 48 h (white bars). Results are expressed as the percentage of cell density value prior to stimulation. Mean+S.D. of three independent experiments are shown, * P
    Figure Legend Snippet: Ca074Me delays B10-induced cell death and transiently reduces Bid degradation, caspase-3 activation and mitochondrial membrane depolarization. ( A ) U87MG cells were incubated with 18 μ M B10 or DMSO in combination with Ca074Me (10 μ M), E64 (50 μ M) or leupeptin (100 μ M). Cell viability was quantified by crystal violet staining after 24 h (black bars) and 48 h (white bars). Results are expressed as the percentage of cell density value prior to stimulation. Mean+S.D. of three independent experiments are shown, * P

    Techniques Used: Activation Assay, Incubation, Staining

    15) Product Images from "Cytomegalovirus m154 Hinders CD48 Cell-Surface Expression and Promotes Viral Escape from Host Natural Killer Cell Control"

    Article Title: Cytomegalovirus m154 Hinders CD48 Cell-Surface Expression and Promotes Viral Escape from Host Natural Killer Cell Control

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004000

    CD48 undergoes proteolytic degradation during MCMV infection. (A) MEFs and peritoneal macrophages were infected at an moi of 0.025 or 0.1, respectively with wt MCMV and MCMVΔm154. At the indicated days after infection the amount of extracellular (MEFs) or cell-associated (macrophages) infectious virus present in the cultures was determined. Each data point represents the average and standard deviation of three separate cultures. (B) Peritoneal macrophages were mock-infected or infected with wt MCMV at an moi of 10 for 72 h. Whole-cell RNA was harvested, treated with DNase and reverse-transcribed using oligo(dT). PCRs were performed using primer sets specific for murine CD48, m154, and β-actin. The amplified products were separated on agarose gels and visualized with ethidium bromide staining. As shown, specific PCR-amplified products were not detected in control reactions in which the reverse transcriptase (RT) was not added during the reverse transcription reaction. (C) Peritoneal macrophages were mock-infected or infected with wt MCMV at an moi of 10. At the indicated time points after infection, cell lysates were prepared, and subjected to Western blot analysis as indicated in Figure 4B using an anti-mouse CD48 mAb or the anti-m154 mAb followed by anti-Armenian hamster HRP or by anti-mouse IgG HRP, respectively. A mAb anti-β-actin followed by anti-rabbit IgG HRP was used as an internal control. (D) Peritoneal macrophages were mock-infected (mock) or infected with wt MCMV at an moi of 10 in the absence (-) or presence of MG-132 or leupeptin. Cell lysates were prepared at 72 hpi and subjected to Western blot analysis as indicated in C. (E) Peritoneal macrophages were mock-infected or infected with wt MCMV at an moi of 10 for 72 h. When indicated, cultures were treated with MG-132 or leupeptin. Cells were fixed, permeabilized, and stained with anti-CD48-Alexa Fluor 488, and anti-m154 mAb followed by an anti-mouse IgG Alexa fluor 555, and analyzed as indicated in Figure 4C . Shown are representative cells from cultures stained for m154 (panels a, e, i, and m), for CD48 (panels b, f, j, and n), nuclei stained with the DAPI reagent (panels c, g, k, and o), and overlaid images (panels d, h, l, and p). Images were captured at 40× magnification.
    Figure Legend Snippet: CD48 undergoes proteolytic degradation during MCMV infection. (A) MEFs and peritoneal macrophages were infected at an moi of 0.025 or 0.1, respectively with wt MCMV and MCMVΔm154. At the indicated days after infection the amount of extracellular (MEFs) or cell-associated (macrophages) infectious virus present in the cultures was determined. Each data point represents the average and standard deviation of three separate cultures. (B) Peritoneal macrophages were mock-infected or infected with wt MCMV at an moi of 10 for 72 h. Whole-cell RNA was harvested, treated with DNase and reverse-transcribed using oligo(dT). PCRs were performed using primer sets specific for murine CD48, m154, and β-actin. The amplified products were separated on agarose gels and visualized with ethidium bromide staining. As shown, specific PCR-amplified products were not detected in control reactions in which the reverse transcriptase (RT) was not added during the reverse transcription reaction. (C) Peritoneal macrophages were mock-infected or infected with wt MCMV at an moi of 10. At the indicated time points after infection, cell lysates were prepared, and subjected to Western blot analysis as indicated in Figure 4B using an anti-mouse CD48 mAb or the anti-m154 mAb followed by anti-Armenian hamster HRP or by anti-mouse IgG HRP, respectively. A mAb anti-β-actin followed by anti-rabbit IgG HRP was used as an internal control. (D) Peritoneal macrophages were mock-infected (mock) or infected with wt MCMV at an moi of 10 in the absence (-) or presence of MG-132 or leupeptin. Cell lysates were prepared at 72 hpi and subjected to Western blot analysis as indicated in C. (E) Peritoneal macrophages were mock-infected or infected with wt MCMV at an moi of 10 for 72 h. When indicated, cultures were treated with MG-132 or leupeptin. Cells were fixed, permeabilized, and stained with anti-CD48-Alexa Fluor 488, and anti-m154 mAb followed by an anti-mouse IgG Alexa fluor 555, and analyzed as indicated in Figure 4C . Shown are representative cells from cultures stained for m154 (panels a, e, i, and m), for CD48 (panels b, f, j, and n), nuclei stained with the DAPI reagent (panels c, g, k, and o), and overlaid images (panels d, h, l, and p). Images were captured at 40× magnification.

    Techniques Used: Infection, Standard Deviation, Amplification, Staining, Polymerase Chain Reaction, Western Blot

    16) Product Images from "Intraneuronal aggregation of the β-CTF fragment of APP (C99) induces Aβ-independent lysosomal-autophagic pathology"

    Article Title: Intraneuronal aggregation of the β-CTF fragment of APP (C99) induces Aβ-independent lysosomal-autophagic pathology

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-016-1577-6

    In vitro analysis shows that APP-CTFs are degraded by cathepsins through autophagy. a , b SH-APPswe cells were treated with NH 4 Cl, leupeptin (Leu), pepstatin A (PepA) or PADK and analyzed by western blot using α-APPct. Arrows correspond to a non-identified 25 kDa APP-CTF fragment. Bars in b correspond to quantification of C99 and C83 immunoreactivities obtained in a and expressed relative to expressions measured in DMSO-treated cells normalized to actin. Data are represented as mean ± SEM, as determined by ANOVA one-way Dunnett post hoc test, ** p
    Figure Legend Snippet: In vitro analysis shows that APP-CTFs are degraded by cathepsins through autophagy. a , b SH-APPswe cells were treated with NH 4 Cl, leupeptin (Leu), pepstatin A (PepA) or PADK and analyzed by western blot using α-APPct. Arrows correspond to a non-identified 25 kDa APP-CTF fragment. Bars in b correspond to quantification of C99 and C83 immunoreactivities obtained in a and expressed relative to expressions measured in DMSO-treated cells normalized to actin. Data are represented as mean ± SEM, as determined by ANOVA one-way Dunnett post hoc test, ** p

    Techniques Used: In Vitro, Western Blot

    17) Product Images from "WNK4 Diverts the Thiazide-sensitive NaCl Cotransporter to the Lysosome and Stimulates AP-3 Interaction *"

    Article Title: WNK4 Diverts the Thiazide-sensitive NaCl Cotransporter to the Lysosome and Stimulates AP-3 Interaction *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.008185

    WNK4 promotes a generalized increase in total lysosomal content. A , laser-scanning confocal images of HEK-293H cells transiently transfected with either empty vector or Myc-tagged WNK4, pretreated with leupeptin for 14 h. Endogenous LAMP-2 was labeled
    Figure Legend Snippet: WNK4 promotes a generalized increase in total lysosomal content. A , laser-scanning confocal images of HEK-293H cells transiently transfected with either empty vector or Myc-tagged WNK4, pretreated with leupeptin for 14 h. Endogenous LAMP-2 was labeled

    Techniques Used: Transfection, Plasmid Preparation, Labeling

    WNK4 diverts NCC to the lysosomal pathway. A , protein densitometry measurements of HEK-293H cells transiently expressing 2×HA-NCC plus empty vector or 2×HA-NCC plus WNK4, treated in the absence and presence of leupeptin (10 μ m
    Figure Legend Snippet: WNK4 diverts NCC to the lysosomal pathway. A , protein densitometry measurements of HEK-293H cells transiently expressing 2×HA-NCC plus empty vector or 2×HA-NCC plus WNK4, treated in the absence and presence of leupeptin (10 μ m

    Techniques Used: Expressing, Plasmid Preparation

    18) Product Images from "The Lysosomal Trafficking Transmembrane Protein 106B Is Linked to Cell Death *"

    Article Title: The Lysosomal Trafficking Transmembrane Protein 106B Is Linked to Cell Death *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.737171

    TMEM106B-NTF-induced cell death is leupeptin-sensitive. A and B , HeLa cells, seeded on 6-well plates at 5 × 10 4 cells/well, were infected with adenovirus vectors encoding TMEM106B-FL at an MOI of 800 or TMEM106B(1–127) or TMEM106B(1–106) at an MOI of 300. After the infection, cells were co-incubated with (+) or without (−) 50 μg/ml leupeptin. At 18 h after the start of infection, media were replaced with DMEM/N2 supplement containing (+) or not containing (−) 50 μg/ml leupeptin. At 24 h after the replacement of media, LDH release was measured ( A ), and the cells were harvested for immunoblotting analysis ( IB ) using the TMEM106B antibody ( B ). *, p
    Figure Legend Snippet: TMEM106B-NTF-induced cell death is leupeptin-sensitive. A and B , HeLa cells, seeded on 6-well plates at 5 × 10 4 cells/well, were infected with adenovirus vectors encoding TMEM106B-FL at an MOI of 800 or TMEM106B(1–127) or TMEM106B(1–106) at an MOI of 300. After the infection, cells were co-incubated with (+) or without (−) 50 μg/ml leupeptin. At 18 h after the start of infection, media were replaced with DMEM/N2 supplement containing (+) or not containing (−) 50 μg/ml leupeptin. At 24 h after the replacement of media, LDH release was measured ( A ), and the cells were harvested for immunoblotting analysis ( IB ) using the TMEM106B antibody ( B ). *, p

    Techniques Used: Infection, Incubation

    19) Product Images from "Autophagosome immunoisolation from GFP-LC3B mouse tissue"

    Article Title: Autophagosome immunoisolation from GFP-LC3B mouse tissue

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1539591

    Intraperitoneal injection of leupeptin as a method to inhibit autophagy flux. (a) Immunoblot and (b) quantification of the levels of GFP-LC3, LC3-I, LC3-II, and GAPDH in the retina, brain, liver and lung of GFP-LC3 mice with a single intraperitoneal injection of leupeptin versus saline (as control). Samples are from the cell lysates and demonstrate the increase in LC3-II relative to LC3-I in the leupeptin sample. Protein levels are normalized to GAPDH levels. * P
    Figure Legend Snippet: Intraperitoneal injection of leupeptin as a method to inhibit autophagy flux. (a) Immunoblot and (b) quantification of the levels of GFP-LC3, LC3-I, LC3-II, and GAPDH in the retina, brain, liver and lung of GFP-LC3 mice with a single intraperitoneal injection of leupeptin versus saline (as control). Samples are from the cell lysates and demonstrate the increase in LC3-II relative to LC3-I in the leupeptin sample. Protein levels are normalized to GAPDH levels. * P

    Techniques Used: Injection, Mouse Assay

    20) Product Images from "Immuno-evasive tactics by schistosomes identifies an effective allergy preventative"

    Article Title: Immuno-evasive tactics by schistosomes identifies an effective allergy preventative

    Journal: Experimental parasitology

    doi: 10.1016/j.exppara.2015.03.012

    Schistosomal calpain-like protease generates SG-sCD23 A. Leupeptin (5µg/ml) inhibits SEA-mediated reduction of surface CD23 on tonsil MC (18 hr culture). Shown are CD23 levels on monocytes. B. Leupeptin inhibits live schistosomula from reducing surface CD23 on purified tonsil B cells (representative of 5 separate experiments; 18 hr culture; * P
    Figure Legend Snippet: Schistosomal calpain-like protease generates SG-sCD23 A. Leupeptin (5µg/ml) inhibits SEA-mediated reduction of surface CD23 on tonsil MC (18 hr culture). Shown are CD23 levels on monocytes. B. Leupeptin inhibits live schistosomula from reducing surface CD23 on purified tonsil B cells (representative of 5 separate experiments; 18 hr culture; * P

    Techniques Used: Purification

    21) Product Images from "ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-β accumulation"

    Article Title: ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-β accumulation

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.170233

    Reduced lysosomal delivery after Hrs/Tsg101 depletion. (A) H4-APP cells depleted of Hrs (siHrs) or Tsg101 (siTsg101) were treated with or without leupeptin and stained with 6E10 (green) and LAMP1 (red). Scale bars: 5 μm. (B) Quantification of 6E10 colocalization with LAMP1 (using Velocity). Results are mean±s.d. of three experiments. * P
    Figure Legend Snippet: Reduced lysosomal delivery after Hrs/Tsg101 depletion. (A) H4-APP cells depleted of Hrs (siHrs) or Tsg101 (siTsg101) were treated with or without leupeptin and stained with 6E10 (green) and LAMP1 (red). Scale bars: 5 μm. (B) Quantification of 6E10 colocalization with LAMP1 (using Velocity). Results are mean±s.d. of three experiments. * P

    Techniques Used: Staining

    APP traffics through the same MVBs as EGFR. (A) H4-APP cells stimulated with EGF–Alexa-Fluor-488 (EGF-488; green) for 10 or 45 min were stained for APP/Aβ with 6E10 (red). Scale bar: 5 μm. (B) Quantification of APP colocalization with EGF-488. (C) H4-APP cells treated with leupeptin were stained with 6E10 (green), cathepsin D (red) and DAPI (blue). Scale bars: 10 μm. (D) Quantification of 6E10 colocalization with cathepsin D. All results are mean±s.d. ( n =3 separate experiments). * P
    Figure Legend Snippet: APP traffics through the same MVBs as EGFR. (A) H4-APP cells stimulated with EGF–Alexa-Fluor-488 (EGF-488; green) for 10 or 45 min were stained for APP/Aβ with 6E10 (red). Scale bar: 5 μm. (B) Quantification of APP colocalization with EGF-488. (C) H4-APP cells treated with leupeptin were stained with 6E10 (green), cathepsin D (red) and DAPI (blue). Scale bars: 10 μm. (D) Quantification of 6E10 colocalization with cathepsin D. All results are mean±s.d. ( n =3 separate experiments). * P

    Techniques Used: Staining

    22) Product Images from "Cigarette smoke extract modulates Pseudomonas aeruginosa bacterial load via USP25/HDAC11 axis in lung epithelial cells"

    Article Title: Cigarette smoke extract modulates Pseudomonas aeruginosa bacterial load via USP25/HDAC11 axis in lung epithelial cells

    Journal: American journal of physiology. Lung cellular and molecular physiology

    doi: 10.1152/ajplung.00142.2019

    Histone deacetylase 11 (HDAC11) protein was unstable and degraded through proteasome system. A : BEAS-2B cells were treated with the protein synthesis inhibitor cycloheximide (CHX; 40 μM), proteasome inhibitor MG132 (40 μM), or lysosome inhibitor leupeptin (20 μM) at various time points as indicated. Cell lysates were subjected to HDAC11 or GAPDH immunoblotting analysis. Densitometry results are plotted in the lower panel. B : wild-type pcDNA3.1D-His-V5/HDAC11 plasmids were introduced into Beas-2B cells for 48 h. Cell lysates were applied to V5 immunoprecipitation (IP) and the immunoprecipitants were analyzed with ubiquitin and V5 immunoblotting (IB) analysis. Results are representative of n = 3 experiments.
    Figure Legend Snippet: Histone deacetylase 11 (HDAC11) protein was unstable and degraded through proteasome system. A : BEAS-2B cells were treated with the protein synthesis inhibitor cycloheximide (CHX; 40 μM), proteasome inhibitor MG132 (40 μM), or lysosome inhibitor leupeptin (20 μM) at various time points as indicated. Cell lysates were subjected to HDAC11 or GAPDH immunoblotting analysis. Densitometry results are plotted in the lower panel. B : wild-type pcDNA3.1D-His-V5/HDAC11 plasmids were introduced into Beas-2B cells for 48 h. Cell lysates were applied to V5 immunoprecipitation (IP) and the immunoprecipitants were analyzed with ubiquitin and V5 immunoblotting (IB) analysis. Results are representative of n = 3 experiments.

    Techniques Used: Histone Deacetylase Assay, Immunoprecipitation

    23) Product Images from "Mislocalization of phosphotransferase as a cause of mucolipidosis III αβ"

    Article Title: Mislocalization of phosphotransferase as a cause of mucolipidosis III αβ

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

    doi: 10.1073/pnas.1401417111

    K4Q and S15Y αβ phosphotransferase show increased degradation in lysosomes. ( A ) Confocal immunofluorescence microscopy on HeLa cells expressing WT, K4Q, or S15Y αβ phosphotransferase. The cells were incubated with transferrin-Alexa Fluor 488 for 30 min and stained with anti-α subunit. K4Q and S15Y phosphotransferase show substantial colocalization with transferrin-positive endosomes. (Scale bars, 20 μm.) ( B ) Western blot (anti-V5) of HEK293 cells expressing WT, K4Q, or S15Y αβ phosphotransferase treated with or without 100 μM leupeptin and 100 μM pepstatin A for 7 h (representative blot of 2 independent experiments). The levels of the mature β subunits of the mutants increase approximately twofold after inhibition of lysosomal degradation. GAPDH shows equal protein loading.
    Figure Legend Snippet: K4Q and S15Y αβ phosphotransferase show increased degradation in lysosomes. ( A ) Confocal immunofluorescence microscopy on HeLa cells expressing WT, K4Q, or S15Y αβ phosphotransferase. The cells were incubated with transferrin-Alexa Fluor 488 for 30 min and stained with anti-α subunit. K4Q and S15Y phosphotransferase show substantial colocalization with transferrin-positive endosomes. (Scale bars, 20 μm.) ( B ) Western blot (anti-V5) of HEK293 cells expressing WT, K4Q, or S15Y αβ phosphotransferase treated with or without 100 μM leupeptin and 100 μM pepstatin A for 7 h (representative blot of 2 independent experiments). The levels of the mature β subunits of the mutants increase approximately twofold after inhibition of lysosomal degradation. GAPDH shows equal protein loading.

    Techniques Used: Immunofluorescence, Microscopy, Expressing, Incubation, Staining, Western Blot, Inhibition

    24) Product Images from "Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC, Microtubules and Lysosomal/Proteasomal Degradation"

    Article Title: Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC, Microtubules and Lysosomal/Proteasomal Degradation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108570

    Desmosomal proteins are degraded by lysosomes and proteasomes, and co-localise with the lysosomal marker lamp1. (A) Internalised desmosomal proteins are gradually degraded. Western blots of whole MDCK cell lysates following LCM treatment for 0, 8, 16 or 24 hours show that Dsg2, DP and PG were gradually degraded. (B–D) Lysosomal and proteasomal degradation. Cells were treated with LCM for 16 or 24 hours in the presence of the noted inhibitors or vehicle alone (DMS0 or water). Western blots of whole cell lysates show that the lysosomal inhibitors bafilomycin A1 (250 nM), chloroquine (100 µM) and leupeptin (100 µM) and the proteasomal inhibitor MG132 (10 µM) inhibited degradation of Dsg2 (B) and chloroquine and bafilomycin A1 inhibited LCM-induced Dsg3 and PG degradation (C, D). (E) Internalised Dsg2 co-localises with the lysosomal marker lamp1. Cells cultured in NCM or treated with LCM for 16 hours stained for Dsg2 (red) and Lamp1 (green). Co-localisation in the latter cells is indicated by white arrows.Yellow arrows indicate XZ axis. Bar, 5 µm. Fluorescence profile depicts the intensity of staining along the white line in the image. (F) DP degradation was not inhibited by lysosomal inihibitors, but instead by the proteasomal inhibitors bortezomib (20–200 nM) and MG132 (10 µM) (dashed lines indicate lanes which have been re-ordered from the same western blot). Bortizomib had no effect on PG or Dsg2 degradation (F). (G) Western blots of whole cell lysates co-treated with LCM and nocodazole (33 µM) for 16 or 24 hrs shows degradation of DP is unaltered, whilst degradation of PG and Dsg2 is partially inhibited. Bar, 5 µm.
    Figure Legend Snippet: Desmosomal proteins are degraded by lysosomes and proteasomes, and co-localise with the lysosomal marker lamp1. (A) Internalised desmosomal proteins are gradually degraded. Western blots of whole MDCK cell lysates following LCM treatment for 0, 8, 16 or 24 hours show that Dsg2, DP and PG were gradually degraded. (B–D) Lysosomal and proteasomal degradation. Cells were treated with LCM for 16 or 24 hours in the presence of the noted inhibitors or vehicle alone (DMS0 or water). Western blots of whole cell lysates show that the lysosomal inhibitors bafilomycin A1 (250 nM), chloroquine (100 µM) and leupeptin (100 µM) and the proteasomal inhibitor MG132 (10 µM) inhibited degradation of Dsg2 (B) and chloroquine and bafilomycin A1 inhibited LCM-induced Dsg3 and PG degradation (C, D). (E) Internalised Dsg2 co-localises with the lysosomal marker lamp1. Cells cultured in NCM or treated with LCM for 16 hours stained for Dsg2 (red) and Lamp1 (green). Co-localisation in the latter cells is indicated by white arrows.Yellow arrows indicate XZ axis. Bar, 5 µm. Fluorescence profile depicts the intensity of staining along the white line in the image. (F) DP degradation was not inhibited by lysosomal inihibitors, but instead by the proteasomal inhibitors bortezomib (20–200 nM) and MG132 (10 µM) (dashed lines indicate lanes which have been re-ordered from the same western blot). Bortizomib had no effect on PG or Dsg2 degradation (F). (G) Western blots of whole cell lysates co-treated with LCM and nocodazole (33 µM) for 16 or 24 hrs shows degradation of DP is unaltered, whilst degradation of PG and Dsg2 is partially inhibited. Bar, 5 µm.

    Techniques Used: Marker, Western Blot, Laser Capture Microdissection, Cell Culture, Staining, Fluorescence

    25) Product Images from "Porphyromonas gingivalis induce apoptosis in human gingival epithelial cells through a gingipain-dependent mechanism"

    Article Title: Porphyromonas gingivalis induce apoptosis in human gingival epithelial cells through a gingipain-dependent mechanism

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-9-107

    TUNEL assay to detect DNA fragmentation by confocal microscopy . Images are fluorescent confocal staining at ×600 magnification. Negative control was unchallenged HGECs at 24 h (A). Positive control was HGECs treated with DNase 1000 U/ml (B). HGECs were challenged with filtered supernatant of P. gingivalis 33277 culture (C) for 24 h. Additional plates (D to F) show challenge with live P. gingivalis 33277 supernatant pretreated with leupeptin, a selective Rgp inhibitor (D), zFKck, a selective Kgp inhibitor (E), or a cocktail of both inhibitors to inhibit total gingipain activity (F). Challenge for 24 hours with filtered culture supernatant derived from the RgpA/RgpB mutant E8 (G), the Kgp mutant K1A (H) or the RgpA/RgpB/Kgp mutant KDP128 (I), are also shown.
    Figure Legend Snippet: TUNEL assay to detect DNA fragmentation by confocal microscopy . Images are fluorescent confocal staining at ×600 magnification. Negative control was unchallenged HGECs at 24 h (A). Positive control was HGECs treated with DNase 1000 U/ml (B). HGECs were challenged with filtered supernatant of P. gingivalis 33277 culture (C) for 24 h. Additional plates (D to F) show challenge with live P. gingivalis 33277 supernatant pretreated with leupeptin, a selective Rgp inhibitor (D), zFKck, a selective Kgp inhibitor (E), or a cocktail of both inhibitors to inhibit total gingipain activity (F). Challenge for 24 hours with filtered culture supernatant derived from the RgpA/RgpB mutant E8 (G), the Kgp mutant K1A (H) or the RgpA/RgpB/Kgp mutant KDP128 (I), are also shown.

    Techniques Used: TUNEL Assay, Confocal Microscopy, Staining, Negative Control, Positive Control, Activity Assay, Derivative Assay, Mutagenesis

    TUNEL assay to detect DNA fragmentation by confocal microscopy . Images are fluorescent confocal staining at ×600 magnification. Negative control was unchallenged HGECs (A). Positive control was HGECs treated with DNase 1000 U/ml (B). HGECs were challenged with live (C) and heat-killed (D) P. gingivalis 33277 MOI:100 for 24 h. Challenge with MOI:100 for 4 h and MOI:10 for 4 and 24 h gave no staining (data not shown). Additional plates (E to G) show challenge with live P. gingivalis 33277 at MOI:100 for 24 h that were pretreated with leupeptin, a selective Rgp inhibitor (E), zFKck, a selective Kgp inhibitor (F), or a cocktail of both inhibitors to inhibit total gingipain activity (G). Challenge with P. gingivalis W50 (H), the RgpA/RgpB mutant E8 (I), the Kgp mutant K1A (J) or the RgpA/RgpB/Kgp mutant KDP128 (K), at MOI:100 for 24 h are also shown.
    Figure Legend Snippet: TUNEL assay to detect DNA fragmentation by confocal microscopy . Images are fluorescent confocal staining at ×600 magnification. Negative control was unchallenged HGECs (A). Positive control was HGECs treated with DNase 1000 U/ml (B). HGECs were challenged with live (C) and heat-killed (D) P. gingivalis 33277 MOI:100 for 24 h. Challenge with MOI:100 for 4 h and MOI:10 for 4 and 24 h gave no staining (data not shown). Additional plates (E to G) show challenge with live P. gingivalis 33277 at MOI:100 for 24 h that were pretreated with leupeptin, a selective Rgp inhibitor (E), zFKck, a selective Kgp inhibitor (F), or a cocktail of both inhibitors to inhibit total gingipain activity (G). Challenge with P. gingivalis W50 (H), the RgpA/RgpB mutant E8 (I), the Kgp mutant K1A (J) or the RgpA/RgpB/Kgp mutant KDP128 (K), at MOI:100 for 24 h are also shown.

    Techniques Used: TUNEL Assay, Confocal Microscopy, Staining, Negative Control, Positive Control, Activity Assay, Mutagenesis

    26) Product Images from "Mitochondrial NCKX5 regulates melanosomal biogenesis and pigment production"

    Article Title: Mitochondrial NCKX5 regulates melanosomal biogenesis and pigment production

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.232009

    NCKX activity and functional mitochondria are required for melanosomal pigment production. (A–H) Confocal BF images of melan-a melanocytes incubated with corresponding compounds for 72 hours: DMSO (A); phenylthiourea (PTU, 200 nM) (B); DBZ (30 μM) (C); CGP-37157 (10 μM) (D); CCCP (2 μM) (E); Oligomycin (Oligo, 5 μM) (F); Rotenone (Rot, 0.4 μM) (G); and Leupeptin (Leu, 100 mM) (H). Scale bar: 10 µm. (I) Melanin quantification assays for melanocytes incubated with PTU (200 nM), DBZ (30 μM) and CGP-37157 (10 μM). Melanin content was assayed by measuring optical density at 492 nm (OD 492 ). Assays were performed in triplicate. (J) Melanin quantification assays for melanocytes incubated with PTU (200 nM); CCCP (2 μM); Oligo (5 μM); Rot (0.4 μM); Leu (100 mM). Melanin content was assayed by measuring optical density at 492 nm (OD 492 ). Assays in I and J were performed in triplicate. Data shown are means±s.e.m. of the number of triplicate experiments. n.s., not significant; * P
    Figure Legend Snippet: NCKX activity and functional mitochondria are required for melanosomal pigment production. (A–H) Confocal BF images of melan-a melanocytes incubated with corresponding compounds for 72 hours: DMSO (A); phenylthiourea (PTU, 200 nM) (B); DBZ (30 μM) (C); CGP-37157 (10 μM) (D); CCCP (2 μM) (E); Oligomycin (Oligo, 5 μM) (F); Rotenone (Rot, 0.4 μM) (G); and Leupeptin (Leu, 100 mM) (H). Scale bar: 10 µm. (I) Melanin quantification assays for melanocytes incubated with PTU (200 nM), DBZ (30 μM) and CGP-37157 (10 μM). Melanin content was assayed by measuring optical density at 492 nm (OD 492 ). Assays were performed in triplicate. (J) Melanin quantification assays for melanocytes incubated with PTU (200 nM); CCCP (2 μM); Oligo (5 μM); Rot (0.4 μM); Leu (100 mM). Melanin content was assayed by measuring optical density at 492 nm (OD 492 ). Assays in I and J were performed in triplicate. Data shown are means±s.e.m. of the number of triplicate experiments. n.s., not significant; * P

    Techniques Used: Activity Assay, Functional Assay, Incubation

    27) Product Images from "The Carboxyl-terminal PDZ Ligand Motif of Chemokine Receptor CXCR2 Modulates Post-endocytic Sorting and Cellular Chemotaxis *"

    Article Title: The Carboxyl-terminal PDZ Ligand Motif of Chemokine Receptor CXCR2 Modulates Post-endocytic Sorting and Cellular Chemotaxis *

    Journal:

    doi: 10.1074/jbc.M804054200

    CXCR2 is degraded in the lysosome. A , lysosomal inhibitors attenuate CXCL8-mediated CXCR2 degradation. HEK293 CXCR2 WT cells were pretreated with cycloheximide, then treated with vehicle alone, chloroquine ( CHLQ ), ammonium chloride ( NH 4 Cl ), or leupeptin
    Figure Legend Snippet: CXCR2 is degraded in the lysosome. A , lysosomal inhibitors attenuate CXCL8-mediated CXCR2 degradation. HEK293 CXCR2 WT cells were pretreated with cycloheximide, then treated with vehicle alone, chloroquine ( CHLQ ), ammonium chloride ( NH 4 Cl ), or leupeptin

    Techniques Used:

    28) Product Images from "Localisation-based imaging of malarial antigens during erythrocyte entry reaffirms a role for AMA1 but not MTRAP in invasion"

    Article Title: Localisation-based imaging of malarial antigens during erythrocyte entry reaffirms a role for AMA1 but not MTRAP in invasion

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.177741

    MTRAP is cleaved irrespective of invasion or egress from the schizont. (A) Immunoblot analysis of a timecourse of tightly synchronised schizonts and early ring samples grown in the presence or absence of inhibitors of egress (E-64) or invasion [heparin (hep)]. Labelling with rM-tail antibodies labels full-length MTRAP (∼75 kDa) and cleavage products (∼38/44 kDa), including the tail stub (∼15 kDa). (B) Immunoblot analysis of tightly synchronised late-schizont- and early-ring-stage parasites grown in the presence or absence of inhibitors of egress [antipain and leupeptin (A+L)] or E-64. Labelling with rM-tsr antisera identifies full-length MTRAP (∼75 kDa) and the cleaved TSR domain (∼25 kDa). rM-tail labelling identified products as in A. (C,D) Immunofluorescence analysis of very late-stage schizonts (C) treated with E-64 to prevent egress or (D) from untreated culture, co-labelled with mAMA1 (1F9) monoclonal antibodies (green) and rM-tail (left), rM-mid (middle) or rM-tsr (right) antibodies (red in respective images). Scale bars: 1 μm.
    Figure Legend Snippet: MTRAP is cleaved irrespective of invasion or egress from the schizont. (A) Immunoblot analysis of a timecourse of tightly synchronised schizonts and early ring samples grown in the presence or absence of inhibitors of egress (E-64) or invasion [heparin (hep)]. Labelling with rM-tail antibodies labels full-length MTRAP (∼75 kDa) and cleavage products (∼38/44 kDa), including the tail stub (∼15 kDa). (B) Immunoblot analysis of tightly synchronised late-schizont- and early-ring-stage parasites grown in the presence or absence of inhibitors of egress [antipain and leupeptin (A+L)] or E-64. Labelling with rM-tsr antisera identifies full-length MTRAP (∼75 kDa) and the cleaved TSR domain (∼25 kDa). rM-tail labelling identified products as in A. (C,D) Immunofluorescence analysis of very late-stage schizonts (C) treated with E-64 to prevent egress or (D) from untreated culture, co-labelled with mAMA1 (1F9) monoclonal antibodies (green) and rM-tail (left), rM-mid (middle) or rM-tsr (right) antibodies (red in respective images). Scale bars: 1 μm.

    Techniques Used: Immunofluorescence

    29) Product Images from "The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)"

    Article Title: The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073296

    CAR shedding is mediated by metalloproteases. (A) U87 CAR cells plated on poly-L-lysine coated plates were pre-incubated for 45 minutes with a variety of protease inhibitors (10 µM pepstatin A, 10 µM leupeptin, 10 µM E64, 250 µM O-phenanthroline, 25 µM TAPI-1) followed by 3 hours of treatment with 1 µM of PMA. None of the treatments were toxic to the cells under these conditions and concentrations of inhibitors. CAR ECD released into conditioned media was detected via Western blot using anti-CAR N-term. antibody 2240. The broad-spectrum metalloprotease inhibitors TAPI-1 and O-phenathroline decreased PMA-stimulated CAR ECD shedding, while the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin had no effect. Also shown are Western blots of full-length CAR from the corresponding cell lysates (anti-CAR C-term. antibody RP291). (B) U87 CAR cells were treated with PMA (1 µM) or DMSO vehicle, in the presence of 25 µM of the broad spectrum metalloprotease inhibitor GM6001 or its negative control. GM6001, but not its negative control, inhibited PMA-stimulated shedding of CAR ECD. (C) U87 CAR cells were incubated for 3 hours with 1 µM PMA along with 10 µg/ml of TIMPs 1, 2 or 3. TIMP1 and TIMP3, but not TIMP2, decreased PMA-mediated ECD shedding of CAR, suggesting that ADAM10 may be a sheddase. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.
    Figure Legend Snippet: CAR shedding is mediated by metalloproteases. (A) U87 CAR cells plated on poly-L-lysine coated plates were pre-incubated for 45 minutes with a variety of protease inhibitors (10 µM pepstatin A, 10 µM leupeptin, 10 µM E64, 250 µM O-phenanthroline, 25 µM TAPI-1) followed by 3 hours of treatment with 1 µM of PMA. None of the treatments were toxic to the cells under these conditions and concentrations of inhibitors. CAR ECD released into conditioned media was detected via Western blot using anti-CAR N-term. antibody 2240. The broad-spectrum metalloprotease inhibitors TAPI-1 and O-phenathroline decreased PMA-stimulated CAR ECD shedding, while the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin had no effect. Also shown are Western blots of full-length CAR from the corresponding cell lysates (anti-CAR C-term. antibody RP291). (B) U87 CAR cells were treated with PMA (1 µM) or DMSO vehicle, in the presence of 25 µM of the broad spectrum metalloprotease inhibitor GM6001 or its negative control. GM6001, but not its negative control, inhibited PMA-stimulated shedding of CAR ECD. (C) U87 CAR cells were incubated for 3 hours with 1 µM PMA along with 10 µg/ml of TIMPs 1, 2 or 3. TIMP1 and TIMP3, but not TIMP2, decreased PMA-mediated ECD shedding of CAR, suggesting that ADAM10 may be a sheddase. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.

    Techniques Used: Incubation, Western Blot, Protease Inhibitor, Negative Control

    30) Product Images from "An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results"

    Article Title: An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results

    Journal: bioRxiv

    doi: 10.1101/835926

    Addition of separate protease inhibitors partly prevents degradation during sonication (A) Western blot showing the presence of Abf1, which was obtained with either addition of a protease inhibitor tablet or addition of separate protease inhibitors (aprotinin, pepstatin, leupeptin and PMSF). Samples before bead beating, after sonication and before sonication are shown. (B) Percent of input DNA recovered of the samples shown in (A). Two replicates were used and the error bars show the distance from the mean. TFC1 and TUB1 were used as background controls (gray), FCF1 and NHX1 are Abf1 targets ( Kasinathan et al , 2014 , green) (C) Western blots showing presence of Cbf1, Mcm1, Reb1 and Sum1. Samples before bead beating, before sonication and after sonication are shown. All the blots shown were incubated with an antibody specific for V5. Tagged Abf1, Cbf1, Mcm1, Reb1 and Sum1 have a mass of approximately 125, kDa, 83 kDa, 76 kDa, 135 kDa and 162 kDa respectively. The locations of these proteins on the blot are indicated on the right side of the corresponding blots. For all blots a crude lysate from a WT-aa strain was used as a negative control. Below all blots is indicated what percentage of the protein was still intact. This was calculated by dividing the signal from the intact protein (upper band) by the total signal in the lane (upper band + all lower bands).
    Figure Legend Snippet: Addition of separate protease inhibitors partly prevents degradation during sonication (A) Western blot showing the presence of Abf1, which was obtained with either addition of a protease inhibitor tablet or addition of separate protease inhibitors (aprotinin, pepstatin, leupeptin and PMSF). Samples before bead beating, after sonication and before sonication are shown. (B) Percent of input DNA recovered of the samples shown in (A). Two replicates were used and the error bars show the distance from the mean. TFC1 and TUB1 were used as background controls (gray), FCF1 and NHX1 are Abf1 targets ( Kasinathan et al , 2014 , green) (C) Western blots showing presence of Cbf1, Mcm1, Reb1 and Sum1. Samples before bead beating, before sonication and after sonication are shown. All the blots shown were incubated with an antibody specific for V5. Tagged Abf1, Cbf1, Mcm1, Reb1 and Sum1 have a mass of approximately 125, kDa, 83 kDa, 76 kDa, 135 kDa and 162 kDa respectively. The locations of these proteins on the blot are indicated on the right side of the corresponding blots. For all blots a crude lysate from a WT-aa strain was used as a negative control. Below all blots is indicated what percentage of the protein was still intact. This was calculated by dividing the signal from the intact protein (upper band) by the total signal in the lane (upper band + all lower bands).

    Techniques Used: Sonication, Western Blot, Protease Inhibitor, Incubation, Negative Control

    31) Product Images from "Glycogen Synthase Kinase-3β Stabilizes the Interleukin (IL)-22 Receptor from Proteasomal Degradation in Murine Lung Epithelia *"

    Article Title: Glycogen Synthase Kinase-3β Stabilizes the Interleukin (IL)-22 Receptor from Proteasomal Degradation in Murine Lung Epithelia *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.551747

    IL-22 receptor degradation under native and stimulated conditions. A , MLE cells were incubated with CHX (10 μg/ml) and MG132 ( MG , 20 μg/ml) or leupeptin ( Leu , 20 μg/ml), and IL-22R immunoblotting was performed with monoclonal anti-IL-22R
    Figure Legend Snippet: IL-22 receptor degradation under native and stimulated conditions. A , MLE cells were incubated with CHX (10 μg/ml) and MG132 ( MG , 20 μg/ml) or leupeptin ( Leu , 20 μg/ml), and IL-22R immunoblotting was performed with monoclonal anti-IL-22R

    Techniques Used: Incubation

    32) Product Images from "Autophagy-Lysosome Pathway in Renal Tubular Epithelial Cells Is Disrupted by Advanced Glycation End Products in Diabetic Nephropathy *"

    Article Title: Autophagy-Lysosome Pathway in Renal Tubular Epithelial Cells Is Disrupted by Advanced Glycation End Products in Diabetic Nephropathy *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.666354

    Autophagosome and lysosome fusion analysis and LC3 turnover assay in vitro . A , Western blot analysis of LC3 and the difference in the LC3-II levels between samples with and without bafilomycin A1 ( Baf A1 ), leupeptin ( LEU ), and chloroquine ( CQ ) was compared
    Figure Legend Snippet: Autophagosome and lysosome fusion analysis and LC3 turnover assay in vitro . A , Western blot analysis of LC3 and the difference in the LC3-II levels between samples with and without bafilomycin A1 ( Baf A1 ), leupeptin ( LEU ), and chloroquine ( CQ ) was compared

    Techniques Used: LC3 Turnover Assay, In Vitro, Western Blot

    33) Product Images from "Expression and Retention of Thymidine Phosphorylase in Cultured Reticulocytes as a Novel Treatment for MNGIE"

    Article Title: Expression and Retention of Thymidine Phosphorylase in Cultured Reticulocytes as a Novel Treatment for MNGIE

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2020.03.029

    Human TP Is Lost during Differentiation via the Ubiquitination Pathway To determine the mechanism responsible for TP degradation, 1 × 10 6 day 14 cTP cells (which correspond to the polychromatic erythroblast stage) were incubated with either 5 μM MG132 or 10 μM leupeptin or left untreated at 37°C for 24 h. (A) Graph shows expression levels measured using flow cytometry before addition of the inhibitors (start) and after incubation with the indicated inhibitors or an equivalent concentration of DMSO vehicle as a control (N = 3 ± SEM). (B) Ribbon structure of the human TP enzyme. The α domain is depicted in blue, and the β domain is depicted in beige. The ubiquitination residues that correspond to the known murine ubiquitination sites within the TP active site are labeled in purple.
    Figure Legend Snippet: Human TP Is Lost during Differentiation via the Ubiquitination Pathway To determine the mechanism responsible for TP degradation, 1 × 10 6 day 14 cTP cells (which correspond to the polychromatic erythroblast stage) were incubated with either 5 μM MG132 or 10 μM leupeptin or left untreated at 37°C for 24 h. (A) Graph shows expression levels measured using flow cytometry before addition of the inhibitors (start) and after incubation with the indicated inhibitors or an equivalent concentration of DMSO vehicle as a control (N = 3 ± SEM). (B) Ribbon structure of the human TP enzyme. The α domain is depicted in blue, and the β domain is depicted in beige. The ubiquitination residues that correspond to the known murine ubiquitination sites within the TP active site are labeled in purple.

    Techniques Used: Incubation, Expressing, Flow Cytometry, Concentration Assay, Labeling

    34) Product Images from "Ectodomain shedding of FLT3 ligand is mediated by TACE 1"

    Article Title: Ectodomain shedding of FLT3 ligand is mediated by TACE 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0801931

    Characterization of FLT3L shedding activity. COS-7 cells transiently expressing AP-FLT3L were incubated with various protease inhibitors (A and B) or MAP kinase inhibitors (C and D) in the presence (B and D) or absence (A and C) of PMA (25 ng/ml). The inhibitors were used at the following concentrations: GM6001 (10 μM), leupeptin (100μM), STI (0.5 mg/ml), pefabloc SC (0.4 mM), E64 (10μM), TIMPs 1-3 (16 nM), U0126 (5 μM) and SB202190 (20 μM). After the incubation, the supernatants were collected and subjected to a colorimetric assay for AP activity as described in Materials and Methods. Each value represents the mean derived from at least three individual experiments; error bars, SD. *, p
    Figure Legend Snippet: Characterization of FLT3L shedding activity. COS-7 cells transiently expressing AP-FLT3L were incubated with various protease inhibitors (A and B) or MAP kinase inhibitors (C and D) in the presence (B and D) or absence (A and C) of PMA (25 ng/ml). The inhibitors were used at the following concentrations: GM6001 (10 μM), leupeptin (100μM), STI (0.5 mg/ml), pefabloc SC (0.4 mM), E64 (10μM), TIMPs 1-3 (16 nM), U0126 (5 μM) and SB202190 (20 μM). After the incubation, the supernatants were collected and subjected to a colorimetric assay for AP activity as described in Materials and Methods. Each value represents the mean derived from at least three individual experiments; error bars, SD. *, p

    Techniques Used: Activity Assay, Expressing, Incubation, Colorimetric Assay, Derivative Assay

    35) Product Images from "Polymer nanoparticles for cross-presentation of exogenous antigens and enhanced cytotoxic T-lymphocyte immune response"

    Article Title: Polymer nanoparticles for cross-presentation of exogenous antigens and enhanced cytotoxic T-lymphocyte immune response

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S110796

    Cross-presentation of PEI-coated PLGA (OVA) NPs. Notes: ( A ) For the cross-presentation assay, DCs were incubated with soluble OVA (20 μg/mL), PLGA (OVA), or PEI-coated PLGA (OVA) NPs (containing OVA at 20 μg/mL) for 6 hours. The cells were stained with antimouse H-2K b bound to the OVA 257–264 peptide (clone 25-D1.16). Expression patterns of H-2K b –OVA 257–264 complex on DCs were assessed by flow cytometry. The numbers inside each dot blot represent the average percentage of 25-D1.16-positive cells. ( B ) CD8-OVA1.3 T cells (10 5 ) were cocultured with DCs (5×10 4 ) treated with soluble OVA (20 μg/mL), PLGA (OVA), or PEI-coated PLGA (OVA) NPs (20 μg/mL OVA) for 18 hours. The culture supernatants were collected and analyzed by ELISA to measure the levels of IL-2. ( C ) CD8-OVA1.3 T cells (10 5 ) were cocultured with DCs that were pretreated with 10 μM MG-132 or 40 μM leupeptin for 1 hour before treatment with OVA peptide (5 μg/mL) or PEI-coated PLGA (OVA) NPs (20 μg/mL OVA). IL-2 levels were measured by ELISA. Graphs represent means ± SD of duplicates. PEP, OVA peptide; NPs, PEI-coated PLGA (OVA) NPs. * P
    Figure Legend Snippet: Cross-presentation of PEI-coated PLGA (OVA) NPs. Notes: ( A ) For the cross-presentation assay, DCs were incubated with soluble OVA (20 μg/mL), PLGA (OVA), or PEI-coated PLGA (OVA) NPs (containing OVA at 20 μg/mL) for 6 hours. The cells were stained with antimouse H-2K b bound to the OVA 257–264 peptide (clone 25-D1.16). Expression patterns of H-2K b –OVA 257–264 complex on DCs were assessed by flow cytometry. The numbers inside each dot blot represent the average percentage of 25-D1.16-positive cells. ( B ) CD8-OVA1.3 T cells (10 5 ) were cocultured with DCs (5×10 4 ) treated with soluble OVA (20 μg/mL), PLGA (OVA), or PEI-coated PLGA (OVA) NPs (20 μg/mL OVA) for 18 hours. The culture supernatants were collected and analyzed by ELISA to measure the levels of IL-2. ( C ) CD8-OVA1.3 T cells (10 5 ) were cocultured with DCs that were pretreated with 10 μM MG-132 or 40 μM leupeptin for 1 hour before treatment with OVA peptide (5 μg/mL) or PEI-coated PLGA (OVA) NPs (20 μg/mL OVA). IL-2 levels were measured by ELISA. Graphs represent means ± SD of duplicates. PEP, OVA peptide; NPs, PEI-coated PLGA (OVA) NPs. * P

    Techniques Used: Incubation, Staining, Expressing, Flow Cytometry, Cytometry, Dot Blot, Enzyme-linked Immunosorbent Assay

    36) Product Images from "Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein"

    Article Title: Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003309

    RSV F requires post endocytic activation. (A). HeLa cells were pretreated with dec-RVKR-CMK, α -PDX or leupeptin at indicated concentration for 1 h before experiment and inhibitors were continuously present during following steps of the experiment. Cells were infected with RSV or RSVΔGΔSH for 6 h before FACS analysis of GFP expressing cells. (B). HeLa cells were infected with RSV or RSVΔGΔSH for 1 h. Virus inoculum was replaced with medium containing 100 µM dec-RVKR-CMK and incubated for 6 before FACS analysis of GFP expressing cells. (C). Versene detached HeLa cells were pretreated with solvent (MOCK), dec-RVKR-CMK or EIPA at indicated concentrations and inhibitors were continuously present during following steps of the experiment. RSV-R18/DiOC or RSVΔGΔSH-R18/DiOC (moi ∼5) was bound to cells at 4°C. Unbound virus was removed and cells were incubated at 37°C for 2 h in the presence of inhibitor. Cells were fixed and the MFI of DiOC fluorescence measured by FACS and normalized to mock no inhibitor controls. (D). RSV F protein (574 aa) is proteolytically processed by furin like protease at the two sites (A aa-106 and B aa-136) to generate disulfide bonds linked F1+F2 and small peptide p27 (aa sequence depicted above). At the N-terminus of F1 is a FP (fusion peptide) and at the C-terminus TM (transmembrane domain), numbers indicate aa position and red underlines specify peptide sequences detected in mass spectrometry. (E). Proteomic analysis of HEp-2 cells and purified RSV particles. The N-terminal sequence of the p27 peptide (F protein) was quantified by a targeted mass spectrometry based on the selected reaction monitoring (SRM). Representative SRM peaks of peptides (left) FMNYTLNNAKKTNVTLSK 3+ and (right) ELPRFMNYTLNNAK 3+ peptides, corresponding to the aa 113–131 and 109–123 of F protein, respectively. Different SRM transitions for a peptide shown in different colors (see supporting information Table S1 ). The bar graphs show the results of the targeted peptide quantitation, presented as the sum of the areas of all the SRM peaks for a given peptide. Where no peptide peak was detectable, noise values were reported as a reference. RT retention time, and Cps counts per second. (F). HeLa cells were pretreated or MOCK treated with dec-RVKR-CMK, α-PDX, or leupeptin at indicated concentration. RSV (input control in the first line) was bound for 1 h in cold (B-binding) or after wash-away unbound virus was internalized for 1.5 h at 37°C before processing (I-internalized). Lysed cell samples were resolved by SDS-PAGE and blots were probed with anti-F1 antibody. (G). RSV was bound for 1 h in cold to HeLa cells; unbound virus wash away and cells were placed at 37°C for indicated times before, lysis, SDS-PAGE, and processing for western blot probed with anti-F1 antibody.
    Figure Legend Snippet: RSV F requires post endocytic activation. (A). HeLa cells were pretreated with dec-RVKR-CMK, α -PDX or leupeptin at indicated concentration for 1 h before experiment and inhibitors were continuously present during following steps of the experiment. Cells were infected with RSV or RSVΔGΔSH for 6 h before FACS analysis of GFP expressing cells. (B). HeLa cells were infected with RSV or RSVΔGΔSH for 1 h. Virus inoculum was replaced with medium containing 100 µM dec-RVKR-CMK and incubated for 6 before FACS analysis of GFP expressing cells. (C). Versene detached HeLa cells were pretreated with solvent (MOCK), dec-RVKR-CMK or EIPA at indicated concentrations and inhibitors were continuously present during following steps of the experiment. RSV-R18/DiOC or RSVΔGΔSH-R18/DiOC (moi ∼5) was bound to cells at 4°C. Unbound virus was removed and cells were incubated at 37°C for 2 h in the presence of inhibitor. Cells were fixed and the MFI of DiOC fluorescence measured by FACS and normalized to mock no inhibitor controls. (D). RSV F protein (574 aa) is proteolytically processed by furin like protease at the two sites (A aa-106 and B aa-136) to generate disulfide bonds linked F1+F2 and small peptide p27 (aa sequence depicted above). At the N-terminus of F1 is a FP (fusion peptide) and at the C-terminus TM (transmembrane domain), numbers indicate aa position and red underlines specify peptide sequences detected in mass spectrometry. (E). Proteomic analysis of HEp-2 cells and purified RSV particles. The N-terminal sequence of the p27 peptide (F protein) was quantified by a targeted mass spectrometry based on the selected reaction monitoring (SRM). Representative SRM peaks of peptides (left) FMNYTLNNAKKTNVTLSK 3+ and (right) ELPRFMNYTLNNAK 3+ peptides, corresponding to the aa 113–131 and 109–123 of F protein, respectively. Different SRM transitions for a peptide shown in different colors (see supporting information Table S1 ). The bar graphs show the results of the targeted peptide quantitation, presented as the sum of the areas of all the SRM peaks for a given peptide. Where no peptide peak was detectable, noise values were reported as a reference. RT retention time, and Cps counts per second. (F). HeLa cells were pretreated or MOCK treated with dec-RVKR-CMK, α-PDX, or leupeptin at indicated concentration. RSV (input control in the first line) was bound for 1 h in cold (B-binding) or after wash-away unbound virus was internalized for 1.5 h at 37°C before processing (I-internalized). Lysed cell samples were resolved by SDS-PAGE and blots were probed with anti-F1 antibody. (G). RSV was bound for 1 h in cold to HeLa cells; unbound virus wash away and cells were placed at 37°C for indicated times before, lysis, SDS-PAGE, and processing for western blot probed with anti-F1 antibody.

    Techniques Used: Activation Assay, Concentration Assay, Infection, FACS, Expressing, Incubation, Fluorescence, Sequencing, Mass Spectrometry, Purification, Quantitation Assay, Binding Assay, SDS Page, Lysis, Western Blot

    37) Product Images from "Human mast cell tryptase stimulates the release of an IL-8-dependent neutrophil chemotactic activity from human umbilical vein endothelial cells (HUVEC)"

    Article Title: Human mast cell tryptase stimulates the release of an IL-8-dependent neutrophil chemotactic activity from human umbilical vein endothelial cells (HUVEC)

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2000.01271.x

    (a) Effect of the protease inhibitor leupeptin on tryptase-induced release of neutrophil chemotactic activity or of adding heparin alone. T, Tryptase (10 mU/ml); Leu, leupeptin (20 μ g/ml); Hep, heparin (4 μ g/ml). (b) Effect of an IL-8 neutralizing antibody on tumour necrosis factor (TNF)- and tryptase-induced neutrophil chemotactic activity. T, Tryptase (10 mU/ml); TNF, TNF (20 U/ml); IL-8ab, IL-8 neutralizing antibody (10 μ g/ml). Results are expressed as the mean ± s.e.m. of three separate experiments performed either in duplicate or in triplicate. * P
    Figure Legend Snippet: (a) Effect of the protease inhibitor leupeptin on tryptase-induced release of neutrophil chemotactic activity or of adding heparin alone. T, Tryptase (10 mU/ml); Leu, leupeptin (20 μ g/ml); Hep, heparin (4 μ g/ml). (b) Effect of an IL-8 neutralizing antibody on tumour necrosis factor (TNF)- and tryptase-induced neutrophil chemotactic activity. T, Tryptase (10 mU/ml); TNF, TNF (20 U/ml); IL-8ab, IL-8 neutralizing antibody (10 μ g/ml). Results are expressed as the mean ± s.e.m. of three separate experiments performed either in duplicate or in triplicate. * P

    Techniques Used: Protease Inhibitor, Activity Assay

    38) Product Images from "Ii Chain Controls the Transport of Major Histocompatibility Complex Class II Molecules to and from Lysosomes"

    Article Title: Ii Chain Controls the Transport of Major Histocompatibility Complex Class II Molecules to and from Lysosomes

    Journal: The Journal of Cell Biology

    doi:

    Selective redistribution of newly synthesized MHC class II molecules to lysosomes in leupeptin-treated cells analyzed by Percoll gradient centrifugation. ( A ) Leupeptin does not affect the density of compartments containing β-hexosaminidase and Tfn-HRP, markers of lysosomes and endosomes, respectively. After a 3-h incubation with or without 2 mM leupeptin, cells were permitted to internalize Tfn coupled to HRP for 30 min at 37°C (in the presence or absence of leupeptin) before homogenization and fractionation on Percoll density gradients. The β-hexosaminidase and HRP activities were determined in individual fractions. High density fractions (bottom of the gradient) contained most of the β-hexosaminidase activity, while Tfn-HRP was mainly present in low density fractions. No difference was observed between leupeptin-treated and control cells. ( B ) Redistribution of newly synthesized MHC class II molecules into high density fractions in leupeptin-treated cells. After a pulse of [ 35 S]methionine and a 1-, 2-, or 4-h chase in the absence ( left ) or presence ( right ) of leupeptin, I-A b –expressing A20 cells were fractionated on Percoll gradients. The membranes in each of the gradient fractions were pelleted by centrifugation, lysed in Triton X-100, and then immunoprecipitated using mAbs to I-A b (Y3P). The samples were analyzed by SDS-PAGE, and bands corresponding to class II β chains were quantified by phosphorimaging and optical densitometry. Ii-p10 intensity was also quantified after the 4-h chase in the presence of leupeptin ( bottom left ). Leupeptin caused a strong redistribution of class II into high density fractions. The majority of Ii-p10 was also found in lysosome-containing fractions. As expected, Ii-p10 was barely detectable in control cells and thus was not shown.
    Figure Legend Snippet: Selective redistribution of newly synthesized MHC class II molecules to lysosomes in leupeptin-treated cells analyzed by Percoll gradient centrifugation. ( A ) Leupeptin does not affect the density of compartments containing β-hexosaminidase and Tfn-HRP, markers of lysosomes and endosomes, respectively. After a 3-h incubation with or without 2 mM leupeptin, cells were permitted to internalize Tfn coupled to HRP for 30 min at 37°C (in the presence or absence of leupeptin) before homogenization and fractionation on Percoll density gradients. The β-hexosaminidase and HRP activities were determined in individual fractions. High density fractions (bottom of the gradient) contained most of the β-hexosaminidase activity, while Tfn-HRP was mainly present in low density fractions. No difference was observed between leupeptin-treated and control cells. ( B ) Redistribution of newly synthesized MHC class II molecules into high density fractions in leupeptin-treated cells. After a pulse of [ 35 S]methionine and a 1-, 2-, or 4-h chase in the absence ( left ) or presence ( right ) of leupeptin, I-A b –expressing A20 cells were fractionated on Percoll gradients. The membranes in each of the gradient fractions were pelleted by centrifugation, lysed in Triton X-100, and then immunoprecipitated using mAbs to I-A b (Y3P). The samples were analyzed by SDS-PAGE, and bands corresponding to class II β chains were quantified by phosphorimaging and optical densitometry. Ii-p10 intensity was also quantified after the 4-h chase in the presence of leupeptin ( bottom left ). Leupeptin caused a strong redistribution of class II into high density fractions. The majority of Ii-p10 was also found in lysosome-containing fractions. As expected, Ii-p10 was barely detectable in control cells and thus was not shown.

    Techniques Used: Synthesized, Gradient Centrifugation, Incubation, Homogenization, Fractionation, Activity Assay, Expressing, Centrifugation, Immunoprecipitation, SDS Page

    Reversal of the leupeptin block results in MHC class II transport from lysosomes to the cell surface. ( A ) Reversibility of the effect of leupeptin on the intracellular retention of I-A b class II molecules. Cells were metabolically labeled for 20 min, and then chased in the continous presence of leupeptin for 4 h. Leupeptin was then removed, and the cells were incubated for 0–24 h. After cell surface biotinylation, the cells were lysed, and total ( left ) or surface ( right ) I-A b molecules were immunoprecipitated sequentially. Upon removal of leupeptin, the total amount of p70 (the Ii-p10–αβ complex) slowly decreased, while the amount of peptide-loaded, SDS-stable compact dimers (“ C ”) increased. Ii-p10 completely disappeared over this time course. After a lag (see B ), compact dimers began to appear at the cell surface. ( B ) Kinetics of compact dimer formation and transport to the cell surface. Bands corresponding to total and surface compact dimers were quantified by phosphorimaging. The lag between formation of compact dimers and their subsequent appearance at the cell surface was 3–4 h.
    Figure Legend Snippet: Reversal of the leupeptin block results in MHC class II transport from lysosomes to the cell surface. ( A ) Reversibility of the effect of leupeptin on the intracellular retention of I-A b class II molecules. Cells were metabolically labeled for 20 min, and then chased in the continous presence of leupeptin for 4 h. Leupeptin was then removed, and the cells were incubated for 0–24 h. After cell surface biotinylation, the cells were lysed, and total ( left ) or surface ( right ) I-A b molecules were immunoprecipitated sequentially. Upon removal of leupeptin, the total amount of p70 (the Ii-p10–αβ complex) slowly decreased, while the amount of peptide-loaded, SDS-stable compact dimers (“ C ”) increased. Ii-p10 completely disappeared over this time course. After a lag (see B ), compact dimers began to appear at the cell surface. ( B ) Kinetics of compact dimer formation and transport to the cell surface. Bands corresponding to total and surface compact dimers were quantified by phosphorimaging. The lag between formation of compact dimers and their subsequent appearance at the cell surface was 3–4 h.

    Techniques Used: Blocking Assay, Metabolic Labelling, Labeling, Incubation, Immunoprecipitation

    Distribution of MHC class II, Ii chain, and lgp-B in leupeptin-treated cells. ( A ) Quantification of MHC class II, Ii, and lgp-B in control and leupeptin-treated cells. The quantifications were carried out on ultrathin cryosections immunogold labeled for Ii chain (anti–Ii cytosolic tail mAb IN1) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum). In each case, 40 cell profiles were analyzed. ( B ) Immunogold localization of Ii chain, lgp-B, and I-A in leupeptin-treated cells. ( Upper panel ) Ultrathin cryosections were double immunogold labeled with the Ii chain antibody IN-1 (anti–Ii cytosolic domain; Ii CYT ) and rabbit anti–I-A cytosolic tail polyclonal antibody. Ii chain accumulates in I-A–positive compartments displaying internal vesicles and electron-dense content. ( Lower panel ) Ultrathin cryosections were double immunogold labeled with the anti–lgp-B mAb GL2A7 and rabbit anti–I-A cytosolic tail polyclonal antibody. Class II molecules were visualized in electron-dense, lgp-B–positive compartments. The size of the gold particles is indicated. Bars, 120 nm.
    Figure Legend Snippet: Distribution of MHC class II, Ii chain, and lgp-B in leupeptin-treated cells. ( A ) Quantification of MHC class II, Ii, and lgp-B in control and leupeptin-treated cells. The quantifications were carried out on ultrathin cryosections immunogold labeled for Ii chain (anti–Ii cytosolic tail mAb IN1) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum) or immunogold labeled for lgp-B (mAb GL2A7) and MHC class II (rabbit anti–I-A cytoplasmic domain serum). In each case, 40 cell profiles were analyzed. ( B ) Immunogold localization of Ii chain, lgp-B, and I-A in leupeptin-treated cells. ( Upper panel ) Ultrathin cryosections were double immunogold labeled with the Ii chain antibody IN-1 (anti–Ii cytosolic domain; Ii CYT ) and rabbit anti–I-A cytosolic tail polyclonal antibody. Ii chain accumulates in I-A–positive compartments displaying internal vesicles and electron-dense content. ( Lower panel ) Ultrathin cryosections were double immunogold labeled with the anti–lgp-B mAb GL2A7 and rabbit anti–I-A cytosolic tail polyclonal antibody. Class II molecules were visualized in electron-dense, lgp-B–positive compartments. The size of the gold particles is indicated. Bars, 120 nm.

    Techniques Used: Labeling

    Intracellular retention of Ii-p10–associated class II molecules. Cells were pulsed for 20 min and chased for the indicated times (h) in the presence or absence of 2 mM leupeptin. At each time point, the cells were surface biotinylated before lysis. Total and cell surface biotinylated class II molecules were sequentially immunoprecipitated using the mAb Y3P and streptavidin-agarose. The samples were then analyzed by SDS-PAGE without boiling before electrophoresis. In untreated control cells ( top ), peptideloaded, 60-kD compact αβ dimers (“ C ”) began to appear both in total lysates and on the surface after 0.5 h of chase. A small amount of the p70 complex of Ii-p10–αβ dimers appeared transiently, beginning also at 0.5 h of chase. In leupeptin-treated cells ( bottom ), p70 began to accumulate in total lysates by 0.5 h, but little was recovered at the plasma membrane. Peptide-loaded compact dimers only began to appear after 2–4 h of chase both in lysates and at the cell surface.
    Figure Legend Snippet: Intracellular retention of Ii-p10–associated class II molecules. Cells were pulsed for 20 min and chased for the indicated times (h) in the presence or absence of 2 mM leupeptin. At each time point, the cells were surface biotinylated before lysis. Total and cell surface biotinylated class II molecules were sequentially immunoprecipitated using the mAb Y3P and streptavidin-agarose. The samples were then analyzed by SDS-PAGE without boiling before electrophoresis. In untreated control cells ( top ), peptideloaded, 60-kD compact αβ dimers (“ C ”) began to appear both in total lysates and on the surface after 0.5 h of chase. A small amount of the p70 complex of Ii-p10–αβ dimers appeared transiently, beginning also at 0.5 h of chase. In leupeptin-treated cells ( bottom ), p70 began to accumulate in total lysates by 0.5 h, but little was recovered at the plasma membrane. Peptide-loaded compact dimers only began to appear after 2–4 h of chase both in lysates and at the cell surface.

    Techniques Used: Lysis, Immunoprecipitation, SDS Page, Electrophoresis

    Fractionation of leupeptin-treated A20 cells by free flow electrophoresis. ( A ) A20 cells were pulse labeled for 20 min and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-A b class II molecules were immunoprecipitated using mAb MKD6. The samples were then analyzed by SDS-PAGE without boiling. The positions of compact dimers (“ C ”), α, β chains, Ii-p10, and a p12 protein of unknown origin are indicated relative to the positions of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. ( Left ) Anode; ( right ) cathode. ( B ) I-A b – expressing A20 cells were pulse labeled for 20 min and chased for 4 h in the presence of leupeptin before fractionation by FFE. The positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded compact dimers, free α and β chains, and Ii-p10 are indicated. ( C ) Positions of marker enzymes for plasma membrane (alkaline phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase) in the FFE profile shown in B. p70 codistributed largely with the lysosomal marker β-hexosaminidase.
    Figure Legend Snippet: Fractionation of leupeptin-treated A20 cells by free flow electrophoresis. ( A ) A20 cells were pulse labeled for 20 min and chased for 2 h in the presence of leupeptin before fractionation by FFE. Membranes collected in each fraction were pelleted by centrifugation and lysed in Triton X-100, and I-A b class II molecules were immunoprecipitated using mAb MKD6. The samples were then analyzed by SDS-PAGE without boiling. The positions of compact dimers (“ C ”), α, β chains, Ii-p10, and a p12 protein of unknown origin are indicated relative to the positions of markers for the major protein peak (plasma membrane), endosomes/lysosomes (β-hexosaminidase), and anodally shifted CIIVcontaining fractions. ( Left ) Anode; ( right ) cathode. ( B ) I-A b – expressing A20 cells were pulse labeled for 20 min and chased for 4 h in the presence of leupeptin before fractionation by FFE. The positions of p70 (Ii-p10–αβ complexes), 60-kD peptide-loaded compact dimers, free α and β chains, and Ii-p10 are indicated. ( C ) Positions of marker enzymes for plasma membrane (alkaline phosphodiesterase) and endosomes/lysosomes (β-hexosaminidase) in the FFE profile shown in B. p70 codistributed largely with the lysosomal marker β-hexosaminidase.

    Techniques Used: Fractionation, Flow Cytometry, Electrophoresis, Labeling, Centrifugation, Immunoprecipitation, SDS Page, Expressing, Marker

    Leupeptin treatment causes MHC class II molecules to accumulate in lgp-containing structures by immunofluorescence microscopy. Control or leupeptintreated (3 h, 2 mM leupeptin) I-A b –expressing A20 cells were fixed, permeabilized, and then stained for MHC class II (FITC, using mAb Y3P, two upper panels ) vs lgp-B (TRITC, using mAb GL2A7, lower two panels ). In control cells, the small amount of intracellular MHC class II was localized to structures that were generally negative for lgp-B. These probably represented CIIVs a nd early endosomes. Leupeptin treatment, however, induced extensive colocalization of class II and lgp-B.
    Figure Legend Snippet: Leupeptin treatment causes MHC class II molecules to accumulate in lgp-containing structures by immunofluorescence microscopy. Control or leupeptintreated (3 h, 2 mM leupeptin) I-A b –expressing A20 cells were fixed, permeabilized, and then stained for MHC class II (FITC, using mAb Y3P, two upper panels ) vs lgp-B (TRITC, using mAb GL2A7, lower two panels ). In control cells, the small amount of intracellular MHC class II was localized to structures that were generally negative for lgp-B. These probably represented CIIVs a nd early endosomes. Leupeptin treatment, however, induced extensive colocalization of class II and lgp-B.

    Techniques Used: Immunofluorescence, Microscopy, Expressing, Staining

    Immunogold localization of MHC class II molecules in leupeptin-treated cells. Ultrathin cryosections were immunogold labeled with the anti–class II mAb M5.114 and protein A–gold (PAG-10). ( A ) In control cells, MHC class II molecules are found on the plasma membrane, in intracellular compartments characterized by the presence of internal membranes. ( B ) In leupeptin-treated cells (18-h treatment), MHC class II molecules were detected on the plasma membrane and accumulate in electron-dense compartments displaying internal membranes. Bars, 120 nm.
    Figure Legend Snippet: Immunogold localization of MHC class II molecules in leupeptin-treated cells. Ultrathin cryosections were immunogold labeled with the anti–class II mAb M5.114 and protein A–gold (PAG-10). ( A ) In control cells, MHC class II molecules are found on the plasma membrane, in intracellular compartments characterized by the presence of internal membranes. ( B ) In leupeptin-treated cells (18-h treatment), MHC class II molecules were detected on the plasma membrane and accumulate in electron-dense compartments displaying internal membranes. Bars, 120 nm.

    Techniques Used: Labeling

    Leupeptin induces an accumulation of SDS- resistant I-A b Ii-p10 complexes. ( A ) Leupeptin induces the accumulation of 10-kD (Ii-p10) and 70-kD (p70) proteins that coprecipitate with I-A b . I-A b –expressing A20 cells were pulsed for 20 min with [ 35 S]methionine and chased at 37°C for the indicated times in the presence or absence of 2 mM leupeptin. After lysis, the I-A b molecules were immunoprecipitated using the Y3P mAb. The samples were not boiled before SDS-PAGE. Labeled class II molecules were not detected before 30 min of chase because the Y3P mAb used for immunoprecipitation does not detect immature αβ dimers complexed with intact Ii chain. ( B ) p70 represents SDS-stable complexes containing class II α and β chains and a 10-kD protein. After a 20-min pulse and 4-h chase with or without 2 mM leupeptin (lanes Lp and C , respectively), class II molecules were immunoprecipitated using the Y3P mAb, and the samples were boiled ( B ) or not boiled ( NB ) before SDSPAGE. After boiling, p70 dissociated quantitatively into monomers corresponding to αβ and Ii-p10. ( C ) p70 represents SDS-stable I-A b αβ–Ii-p10 complexes. After a 20-min pulse and 4-h chase in the presence of leupeptin, class II molecules were immunoprecipitated with either anti–I-A b (Y3P) or anti–Ii chain cytoplasmic domain (IN-1) mAbs. While both antibodies precipitated the p70 complex, only anti–class II mAb precipitated the 60-kD SDS-stable compact dimer. Thus, p70 but not compact dimers are complexed with Ii chain or Ii chain fragments (i.e., Ii-p10) that contain the Ii chain cytoplasmic domain. ( D ) Kinetics of association between Ii-p10 and I-A b or I-A d . Pulse-chase experiments were performed as above using A20 cells expressing only I-A d or expressing both I-A d and I-A b . I-A d or I-A b –containing complexes were then immunoprecipitated using specific mAbs (Y3P and MKD6, respectively), and the amounts of Ii-p10 associated to the class II molecules were quantified by phosphorimaging. The association of Ii-p10 with I-A b persisted throughout the chase period, while Ii-p10–I-A d complexes appeared only transiently.
    Figure Legend Snippet: Leupeptin induces an accumulation of SDS- resistant I-A b Ii-p10 complexes. ( A ) Leupeptin induces the accumulation of 10-kD (Ii-p10) and 70-kD (p70) proteins that coprecipitate with I-A b . I-A b –expressing A20 cells were pulsed for 20 min with [ 35 S]methionine and chased at 37°C for the indicated times in the presence or absence of 2 mM leupeptin. After lysis, the I-A b molecules were immunoprecipitated using the Y3P mAb. The samples were not boiled before SDS-PAGE. Labeled class II molecules were not detected before 30 min of chase because the Y3P mAb used for immunoprecipitation does not detect immature αβ dimers complexed with intact Ii chain. ( B ) p70 represents SDS-stable complexes containing class II α and β chains and a 10-kD protein. After a 20-min pulse and 4-h chase with or without 2 mM leupeptin (lanes Lp and C , respectively), class II molecules were immunoprecipitated using the Y3P mAb, and the samples were boiled ( B ) or not boiled ( NB ) before SDSPAGE. After boiling, p70 dissociated quantitatively into monomers corresponding to αβ and Ii-p10. ( C ) p70 represents SDS-stable I-A b αβ–Ii-p10 complexes. After a 20-min pulse and 4-h chase in the presence of leupeptin, class II molecules were immunoprecipitated with either anti–I-A b (Y3P) or anti–Ii chain cytoplasmic domain (IN-1) mAbs. While both antibodies precipitated the p70 complex, only anti–class II mAb precipitated the 60-kD SDS-stable compact dimer. Thus, p70 but not compact dimers are complexed with Ii chain or Ii chain fragments (i.e., Ii-p10) that contain the Ii chain cytoplasmic domain. ( D ) Kinetics of association between Ii-p10 and I-A b or I-A d . Pulse-chase experiments were performed as above using A20 cells expressing only I-A d or expressing both I-A d and I-A b . I-A d or I-A b –containing complexes were then immunoprecipitated using specific mAbs (Y3P and MKD6, respectively), and the amounts of Ii-p10 associated to the class II molecules were quantified by phosphorimaging. The association of Ii-p10 with I-A b persisted throughout the chase period, while Ii-p10–I-A d complexes appeared only transiently.

    Techniques Used: Expressing, Lysis, Immunoprecipitation, SDS Page, Labeling, Pulse Chase

    39) Product Images from "Identification of Antimalarial Compounds That Require CLAG3 for Their Uptake by Plasmodium falciparum-Infected Erythrocytes"

    Article Title: Identification of Antimalarial Compounds That Require CLAG3 for Their Uptake by Plasmodium falciparum-Infected Erythrocytes

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00052-19

    Changes in CLAG3 expression in cultures selected with different drugs. (A) Changes in the transcript levels of clag3.1 and clag3.2 in cultures selected with blasticidin S (BS), fosmidomycin (FOSMI), doxycycline (DOXY), leupeptin (LEUP), T3, and T16 compared to unselected control cultures maintained in parallel. The 10G or 3D7-A lines were used for these experiments, as indicated. Cultures selected with DOXY were maintained with the drug at 250 nM for 14 weeks or at 250 nM for 3 weeks and then at 500 nM for 7 additional weeks. Cultures selected with LEUP were first selected at 2.5 μM for 2 weeks and then maintained one additional week at the same concentration (for a total of 3 weeks at 2.5 μM) or one additional week at 5 μM and one week at 8 μM (2.5-5-8 μM bars). Transcript levels are normalized against rhoph2 , which has temporal expression dynamics similar to clag3 genes along the asexual cycle. Values are the log 2 of the normalized expression fold change in drug-selected cultures versus cultures maintained in parallel in the absence of drug. Zero indicates the same expression in selected and control cultures, whereas positive values reflect an increase of expression in drug-selected cultures and negative values reflect reduced expression in drug-selected cultures. Individual values are the average of reactions performed in triplicate, but for each drug selection the result of independent biological samples collected at different times (indicated in weeks) is presented. (B) Resistance of late-stage parasites (pigmented trophozoites and schizonts) to treatment with sorbitol in 10G cultures selected with T3 (10G-T3) or unselected 10G cultures maintained in parallel (10G). “Control” are the same cultures before sorbitol treatment. (C) IFA analysis of mature schizont-infected erythrocytes with anti-CLAG3 and anti-AMA1 antibodies in T3-selected (10G-T3) or unselected 10G cultures. DAPI marks parasite nuclei. Anti-AMA1 antibodies were used to identify schizonts sufficiently mature for CLAG3 expression. The proportion of AMA1-positive infected erythrocytes that are negative for CLAG3 in 10G-T3 or unselected 10G cultures is shown. Values in the bar chart are averages of two independent biological replicates, along with the SD.
    Figure Legend Snippet: Changes in CLAG3 expression in cultures selected with different drugs. (A) Changes in the transcript levels of clag3.1 and clag3.2 in cultures selected with blasticidin S (BS), fosmidomycin (FOSMI), doxycycline (DOXY), leupeptin (LEUP), T3, and T16 compared to unselected control cultures maintained in parallel. The 10G or 3D7-A lines were used for these experiments, as indicated. Cultures selected with DOXY were maintained with the drug at 250 nM for 14 weeks or at 250 nM for 3 weeks and then at 500 nM for 7 additional weeks. Cultures selected with LEUP were first selected at 2.5 μM for 2 weeks and then maintained one additional week at the same concentration (for a total of 3 weeks at 2.5 μM) or one additional week at 5 μM and one week at 8 μM (2.5-5-8 μM bars). Transcript levels are normalized against rhoph2 , which has temporal expression dynamics similar to clag3 genes along the asexual cycle. Values are the log 2 of the normalized expression fold change in drug-selected cultures versus cultures maintained in parallel in the absence of drug. Zero indicates the same expression in selected and control cultures, whereas positive values reflect an increase of expression in drug-selected cultures and negative values reflect reduced expression in drug-selected cultures. Individual values are the average of reactions performed in triplicate, but for each drug selection the result of independent biological samples collected at different times (indicated in weeks) is presented. (B) Resistance of late-stage parasites (pigmented trophozoites and schizonts) to treatment with sorbitol in 10G cultures selected with T3 (10G-T3) or unselected 10G cultures maintained in parallel (10G). “Control” are the same cultures before sorbitol treatment. (C) IFA analysis of mature schizont-infected erythrocytes with anti-CLAG3 and anti-AMA1 antibodies in T3-selected (10G-T3) or unselected 10G cultures. DAPI marks parasite nuclei. Anti-AMA1 antibodies were used to identify schizonts sufficiently mature for CLAG3 expression. The proportion of AMA1-positive infected erythrocytes that are negative for CLAG3 in 10G-T3 or unselected 10G cultures is shown. Values in the bar chart are averages of two independent biological replicates, along with the SD.

    Techniques Used: Expressing, Concentration Assay, Selection, Immunofluorescence, Infection

    40) Product Images from "A Pre-Immediate-Early Role for Tegument ICP0 in the Proteasome-Dependent Entry of Herpes Simplex Virus ▿"

    Article Title: A Pre-Immediate-Early Role for Tegument ICP0 in the Proteasome-Dependent Entry of Herpes Simplex Virus ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00267-11

    Effect of protease inhibitors on HSV entry. (A) CHO-nectin-1 cells were treated with 200 μM E-64d, 100 μM CA074, 50 μM leupeptin, 20 μM TLCK, μM 500 μM pepstatin A, 100 μM phosphoramidon, or 20 μM
    Figure Legend Snippet: Effect of protease inhibitors on HSV entry. (A) CHO-nectin-1 cells were treated with 200 μM E-64d, 100 μM CA074, 50 μM leupeptin, 20 μM TLCK, μM 500 μM pepstatin A, 100 μM phosphoramidon, or 20 μM

    Techniques Used:

    Related Articles

    Cell Culture:

    Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma *
    Article Snippet: .. In some experiments, cells were cultured for up to 7 days in medium containing the proteinase inhibitors leupeptin (Sigma, 100 μ m final concentration) and chymostatin (Sigma, 100 μ m final concentration) ( ) to block KLK activity. .. To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFP-V-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

    Concentration Assay:

    Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma *
    Article Snippet: .. In some experiments, cells were cultured for up to 7 days in medium containing the proteinase inhibitors leupeptin (Sigma, 100 μ m final concentration) and chymostatin (Sigma, 100 μ m final concentration) ( ) to block KLK activity. .. To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFP-V-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

    Incubation:

    Article Title: Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis
    Article Snippet: .. Starvation and recovery protocols For macroautophagy studies, two starvation protocols were used: Protocol 1 consisted of complete nutrient deprivation (amino acid/serum starvation), in which WT and Ctns −/− cells were washed briefly in HBSS (Gibco) and media-aspirated, and fresh HBSS was added again, followed by 45 min of incubation at 37°C, in the presence or absence of the lysosomal inhibitors bafilomycin A (LC laboratories, 100 nM), leupeptin (Sigma, 20 μM) or chloroquine (Sigma, 50 μM). .. Protocol 2 was serum starvation only: cells were briefly washed in serum-free DMEM (containing 1× amino acids) and media-aspirated, and fresh serum-free DMEM was added followed by 5 h incubation at 37°C, in the presence or absence of lysosomal inhibitors.

    other:

    Article Title: MEK Inhibition Leads To Lysosome-Mediated Na+/I- Symporter Protein Degradation In Human Breast Cancer Cells
    Article Snippet: Leupeptin and Chloroquine (Sigma) were dissolved in distilled water at 10mM.

    Activity Assay:

    Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma *
    Article Snippet: .. In some experiments, cells were cultured for up to 7 days in medium containing the proteinase inhibitors leupeptin (Sigma, 100 μ m final concentration) and chymostatin (Sigma, 100 μ m final concentration) ( ) to block KLK activity. .. To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFP-V-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

    Blocking Assay:

    Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma *
    Article Snippet: .. In some experiments, cells were cultured for up to 7 days in medium containing the proteinase inhibitors leupeptin (Sigma, 100 μ m final concentration) and chymostatin (Sigma, 100 μ m final concentration) ( ) to block KLK activity. .. To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFP-V-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

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    Millipore trypsin inhibitor leupeptin
    Conditioned medium from intestinal epithelial cells activates dorsal root ganglia (DRG) sensory neurons by the release of trypsin-like serine protease. Effect of the serine protease inhibitor FUT or the trypsin inhibitor <t>leupeptin</t> on the percentage of responsive neurons (A) and on the Fluo-4 (ΔF/F 0 ) measured Ca 2+ levels (B) stimulated by apical or basal supernatants recovered from control or lipopolysaccharide (LPS)-treated Caco-2 cell monolayers. (C) Percentage of sensory neurons dissociated from DRGs of wild-type (wt) or PAR 2 knockout (KO) mice responding to apical or basal supernatants recovered from control or LPS-treated Caco-2 cell monolayers. Data are expressed as mean±SEM and were compared using a one-way analysis of variance followed by Bonferroni's post-test (n=6, 2 wells per condition for this experimentation) **p
    Trypsin Inhibitor Leupeptin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteolysis of Ebola virus GP and inhibition of infection using cathepsin inhibitors. (A) VeroE6 cells were treated with NH 4 Cl (50 mM), <t>leupeptin</t> (10 μM), E64-d (10 μM), Z-LLL-FMK (10 μM), or CA074 (100 μM) for 1 h at 37°C
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    Conditioned medium from intestinal epithelial cells activates dorsal root ganglia (DRG) sensory neurons by the release of trypsin-like serine protease. Effect of the serine protease inhibitor FUT or the trypsin inhibitor leupeptin on the percentage of responsive neurons (A) and on the Fluo-4 (ΔF/F 0 ) measured Ca 2+ levels (B) stimulated by apical or basal supernatants recovered from control or lipopolysaccharide (LPS)-treated Caco-2 cell monolayers. (C) Percentage of sensory neurons dissociated from DRGs of wild-type (wt) or PAR 2 knockout (KO) mice responding to apical or basal supernatants recovered from control or LPS-treated Caco-2 cell monolayers. Data are expressed as mean±SEM and were compared using a one-way analysis of variance followed by Bonferroni's post-test (n=6, 2 wells per condition for this experimentation) **p

    Journal: Gut

    Article Title: Epithelial expression and function of trypsin-3 in irritable bowel syndrome

    doi: 10.1136/gutjnl-2016-312094

    Figure Lengend Snippet: Conditioned medium from intestinal epithelial cells activates dorsal root ganglia (DRG) sensory neurons by the release of trypsin-like serine protease. Effect of the serine protease inhibitor FUT or the trypsin inhibitor leupeptin on the percentage of responsive neurons (A) and on the Fluo-4 (ΔF/F 0 ) measured Ca 2+ levels (B) stimulated by apical or basal supernatants recovered from control or lipopolysaccharide (LPS)-treated Caco-2 cell monolayers. (C) Percentage of sensory neurons dissociated from DRGs of wild-type (wt) or PAR 2 knockout (KO) mice responding to apical or basal supernatants recovered from control or LPS-treated Caco-2 cell monolayers. Data are expressed as mean±SEM and were compared using a one-way analysis of variance followed by Bonferroni's post-test (n=6, 2 wells per condition for this experimentation) **p

    Article Snippet: Supernatants from apical or basal compartments of Caco-2 cells culture, stimulated or not by LPS (Escherichia coli serotype K235, ATCC13027), were pre-incubated with the serine protease inhibitor FUT-175 (Futhan) (50 μg/mL) or the trypsin inhibitor leupeptin (100 μM) (all from Calbiochem) for 15 min. Additional recordings were made from DRG neurons after addition of trypsin-3 (10 nM, R & D) or its vehicle (HBSS+0.025% Brij35) in the presence or absence of specific PAR1 (SCH79797, Tocris Bio-Techne, UK) or PAR4 (ML-354, Tocris Bio-Techne) antagonists (all at 10 µM for 5 min).

    Techniques: Protease Inhibitor, Knock-Out, Mouse Assay

    Proteolysis of Ebola virus GP and inhibition of infection using cathepsin inhibitors. (A) VeroE6 cells were treated with NH 4 Cl (50 mM), leupeptin (10 μM), E64-d (10 μM), Z-LLL-FMK (10 μM), or CA074 (100 μM) for 1 h at 37°C

    Journal:

    Article Title: Proteolysis of the Ebola Virus Glycoproteins Enhances Virus Binding and Infectivity ▿

    doi: 10.1128/JVI.01170-07

    Figure Lengend Snippet: Proteolysis of Ebola virus GP and inhibition of infection using cathepsin inhibitors. (A) VeroE6 cells were treated with NH 4 Cl (50 mM), leupeptin (10 μM), E64-d (10 μM), Z-LLL-FMK (10 μM), or CA074 (100 μM) for 1 h at 37°C

    Article Snippet: VeroE6 cells were pretreated for 1 h with 50 mM ammonium chloride, 10 μM leupeptin, 10 μM E64d (EMD Biosciences), 10 μM Z-Leu-Leu-Leu-fluoromethyl ketone (Z-LLL-FMK; EMD Biosciences), or 100 μM CA074 (EMD Biosciences).

    Techniques: Inhibition, Infection

    Palmitoylation protects the Fas receptor from lysosomal degradation. ( a ) The lysosomal degradation pathway inhibitors Leupeptin (100 and 300 μ M), chloroquine (50 and 100 μ M) and bafilomycin A1 (30, 100 and 300 nM)

    Journal: Cell Death and Differentiation

    Article Title: Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability

    doi: 10.1038/cdd.2014.153

    Figure Lengend Snippet: Palmitoylation protects the Fas receptor from lysosomal degradation. ( a ) The lysosomal degradation pathway inhibitors Leupeptin (100 and 300 μ M), chloroquine (50 and 100 μ M) and bafilomycin A1 (30, 100 and 300 nM)

    Article Snippet: In order to inhibit the lysosomal degradation pathway, chloroquine (Sigma), bafilomycine A1 (Calbiochem, Gibbstown, NJ, USA) or leupeptin (Calbiochem) were added to the culture medium for 16 h. Protein synthesis inhibition was studied by adding cycloheximide (100 μ g/ml; Sigma) to the culture medium.

    Techniques:

    Schistosomal calpain-like protease generates SG-sCD23 A. Leupeptin (5µg/ml) inhibits SEA-mediated reduction of surface CD23 on tonsil MC (18 hr culture). Shown are CD23 levels on monocytes. B. Leupeptin inhibits live schistosomula from reducing surface CD23 on purified tonsil B cells (representative of 5 separate experiments; 18 hr culture; * P

    Journal: Experimental parasitology

    Article Title: Immuno-evasive tactics by schistosomes identifies an effective allergy preventative

    doi: 10.1016/j.exppara.2015.03.012

    Figure Lengend Snippet: Schistosomal calpain-like protease generates SG-sCD23 A. Leupeptin (5µg/ml) inhibits SEA-mediated reduction of surface CD23 on tonsil MC (18 hr culture). Shown are CD23 levels on monocytes. B. Leupeptin inhibits live schistosomula from reducing surface CD23 on purified tonsil B cells (representative of 5 separate experiments; 18 hr culture; * P

    Article Snippet: Protease inhibitors including, leupeptin, CA-074, and cathepsin L inhibitor, were used in cultures at 5 µg/ml (Calbiochem-EMD Millipore, Billerica, MA).

    Techniques: Purification