Structured Review

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

1) Product Images from "A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation"

Article Title: A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.155234

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

Techniques Used: Activity Assay

2) Product Images from "Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling"

Article Title: Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-02-0100

Complex formation of PLD1 with caveolin-1 and with the EGFR in the CEM. COS-7 cells transfected with WT or C240S/C241S mutant of PLD1 were incubated in the presence or absence of 100 nM of EGF for 0.5 min. The CEM fraction was prepared, and the proteins were solubilized in an extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Nonidet P-40, 0.4% deoxycholate, 60 mM β-octylglucopyranoside, 0.5 mM phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, and 5 μg/ml aprotinin). The sample was centrifuged at 150,000 × g for 1 h, and the solubilized supernatant was incubated with anti-PLD antibody precoupled to protein A agarose resin. The immune complexes were subjected to 6–16% gradient SDS-PAGE followed by immunoblot analysis with anti-PLD, anti-EGFR, or anti-caveolin-1 antibody. The blots shown are representative of three separate results. I.P., immunoprecipitate.
Figure Legend Snippet: Complex formation of PLD1 with caveolin-1 and with the EGFR in the CEM. COS-7 cells transfected with WT or C240S/C241S mutant of PLD1 were incubated in the presence or absence of 100 nM of EGF for 0.5 min. The CEM fraction was prepared, and the proteins were solubilized in an extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Nonidet P-40, 0.4% deoxycholate, 60 mM β-octylglucopyranoside, 0.5 mM phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, and 5 μg/ml aprotinin). The sample was centrifuged at 150,000 × g for 1 h, and the solubilized supernatant was incubated with anti-PLD antibody precoupled to protein A agarose resin. The immune complexes were subjected to 6–16% gradient SDS-PAGE followed by immunoblot analysis with anti-PLD, anti-EGFR, or anti-caveolin-1 antibody. The blots shown are representative of three separate results. I.P., immunoprecipitate.

Techniques Used: Transfection, Mutagenesis, Incubation, SDS Page

3) Product Images from "Tetraspanin Proteins Regulate Membrane Type-1 Matrix Metalloproteinase-dependent Pericellular Proteolysis"

Article Title: Tetraspanin Proteins Regulate Membrane Type-1 Matrix Metalloproteinase-dependent Pericellular Proteolysis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E08-11-1149

Tetraspanin effects on MT1-MMP-dependent fibronectin degradation. MCF-7-VC and MCF-7-MT1 cells were treated with siRNAs, plated 4 d later on fibronectin (FN)-coated slides, and then incubated overnight in serum-free media with protease inhibitors (50 μg/ml aprotinin, 2 μM leupeptin, 20 μM E64, and 20 μM pepstatin A) to prevent non-MMP proteolysis of the FN. The following day, cells were washed, fixed, and FN was detected using an anti-FN antibody followed by Alexa 488-conjugated anti-mouse secondary antibody (green). The position of the cells was determined by staining the cytoskeleton with Alexa Fluor 546-phalloidin (red) and the nuclei with DAPI (blue). Representative fluorescent images (merge green/red/blue or green only) are shown. Bar, 20 μm. For the bar graph, 100% fibronectin degradation is defined as 0 pixel density within defined proteolyzed areas, whereas 0% degradation is pixel density in the unperturbed fibronectin layer. Results are mean of percentage of fibronectin degradation ± SD (n = 3; *p
Figure Legend Snippet: Tetraspanin effects on MT1-MMP-dependent fibronectin degradation. MCF-7-VC and MCF-7-MT1 cells were treated with siRNAs, plated 4 d later on fibronectin (FN)-coated slides, and then incubated overnight in serum-free media with protease inhibitors (50 μg/ml aprotinin, 2 μM leupeptin, 20 μM E64, and 20 μM pepstatin A) to prevent non-MMP proteolysis of the FN. The following day, cells were washed, fixed, and FN was detected using an anti-FN antibody followed by Alexa 488-conjugated anti-mouse secondary antibody (green). The position of the cells was determined by staining the cytoskeleton with Alexa Fluor 546-phalloidin (red) and the nuclei with DAPI (blue). Representative fluorescent images (merge green/red/blue or green only) are shown. Bar, 20 μm. For the bar graph, 100% fibronectin degradation is defined as 0 pixel density within defined proteolyzed areas, whereas 0% degradation is pixel density in the unperturbed fibronectin layer. Results are mean of percentage of fibronectin degradation ± SD (n = 3; *p

Techniques Used: Incubation, Staining

4) Product Images from "Human Plasmacytoid Dendritic Cells Efficiently Capture HIV-1 Envelope Glycoproteins via CD4 for Antigen Presentation"

Article Title: Human Plasmacytoid Dendritic Cells Efficiently Capture HIV-1 Envelope Glycoproteins via CD4 for Antigen Presentation

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

doi: 10.4049/jimmunol.1202489

Both CLR-mediated and CD4-mediated endocytosis of Env result in similar kinetics of antigen processing Cells were incubated with 10µg/mL FITC-labeled Env or HA for 90 min at 4°C or 37°C, then washed twice and cultured at 4°C or 37°C for up to 24h. In some cases, MDCs were pre-incubated and the experiment carried out in the presence of 1mM leupeptin (Leu) to inhibit proteolytic degradation. Samples were then stained with DC markers for analysis by flow cytometry and gated on HLA-DR + /CD11c + (MDCs), CD123 + (PDCs), CD1a + (LCs) or HLA-DR + (DDCs). A) Representative MDC donor showing the percentage of positive cells. B) The MFI for each timepoint was normalised to the 0 min 37°C control for each Ag and the data shown here are the mean ± SD for for 3–5 donors for Env and 1 donor for HA. C) Alternatively, MDCs were incubated with Env in the presence of mannan or incubated with Env-D368R to isolate degradation via CD4 and CLR-mediated uptake pathways respectively. Leupeptin was also included in these experiments as a control. Data here are the mean ± SD of 2 donors.
Figure Legend Snippet: Both CLR-mediated and CD4-mediated endocytosis of Env result in similar kinetics of antigen processing Cells were incubated with 10µg/mL FITC-labeled Env or HA for 90 min at 4°C or 37°C, then washed twice and cultured at 4°C or 37°C for up to 24h. In some cases, MDCs were pre-incubated and the experiment carried out in the presence of 1mM leupeptin (Leu) to inhibit proteolytic degradation. Samples were then stained with DC markers for analysis by flow cytometry and gated on HLA-DR + /CD11c + (MDCs), CD123 + (PDCs), CD1a + (LCs) or HLA-DR + (DDCs). A) Representative MDC donor showing the percentage of positive cells. B) The MFI for each timepoint was normalised to the 0 min 37°C control for each Ag and the data shown here are the mean ± SD for for 3–5 donors for Env and 1 donor for HA. C) Alternatively, MDCs were incubated with Env in the presence of mannan or incubated with Env-D368R to isolate degradation via CD4 and CLR-mediated uptake pathways respectively. Leupeptin was also included in these experiments as a control. Data here are the mean ± SD of 2 donors.

Techniques Used: Incubation, Labeling, Cell Culture, Staining, Flow Cytometry, Cytometry

5) Product Images from "Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor"

Article Title: Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200312055

Lysosomal degradation of the CI-MPR upon depletion of retromer. (A) Immunoblot analysis of CI-MPR half-life in mock- and hVps26-siRNA–treated HeLa cells after treatment with 40 μg/ml cycloheximide. Tubulin was used as a control. (B) Immunoblot analysis of CI-MPR levels after silencing hVps26. HeLa cells were incubated in the absence or presence of the lysosomal inhibitors leupeptin (Leup, 1 mg/ml), E64 (5 μg/ml), and methionine methyl ester (MME, 10 mM) for 3 h, as indicated in the figure. Blots were probed with antibodies to the CI-MPR or tubulin (control). (C–K) Immunofluorescence microscopy of mock-treated (C–E) or hVps26-siRNA–treated HeLa cells (F–K) incubated in the absence or presence of the lysosomal inhibitors described in A, and stained with the antibody to the CI-MPR and donkey Cy3-conjugated anti–mouse Igs. Lamp-1-YFP (I–K) was expressed by transient transfection. In this case, hVps26-depleted cells were treated with MME/Leup/E64 protease inhibitors. Arrowheads in I–K indicate colocalization of Lamp-1-YFP with CI-MPR. Bar, 10 μm.
Figure Legend Snippet: Lysosomal degradation of the CI-MPR upon depletion of retromer. (A) Immunoblot analysis of CI-MPR half-life in mock- and hVps26-siRNA–treated HeLa cells after treatment with 40 μg/ml cycloheximide. Tubulin was used as a control. (B) Immunoblot analysis of CI-MPR levels after silencing hVps26. HeLa cells were incubated in the absence or presence of the lysosomal inhibitors leupeptin (Leup, 1 mg/ml), E64 (5 μg/ml), and methionine methyl ester (MME, 10 mM) for 3 h, as indicated in the figure. Blots were probed with antibodies to the CI-MPR or tubulin (control). (C–K) Immunofluorescence microscopy of mock-treated (C–E) or hVps26-siRNA–treated HeLa cells (F–K) incubated in the absence or presence of the lysosomal inhibitors described in A, and stained with the antibody to the CI-MPR and donkey Cy3-conjugated anti–mouse Igs. Lamp-1-YFP (I–K) was expressed by transient transfection. In this case, hVps26-depleted cells were treated with MME/Leup/E64 protease inhibitors. Arrowheads in I–K indicate colocalization of Lamp-1-YFP with CI-MPR. Bar, 10 μm.

Techniques Used: Incubation, Immunofluorescence, Microscopy, Staining, Transfection

6) Product Images from "The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin"

Article Title: The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin

Journal: Matrix biology : journal of the International Society for Matrix Biology

doi: 10.1016/j.matbio.2017.04.001

Sortilin levels are stabilized by lysosomal inhibition. Serum-starved PC3/GFP-Sortilin cells were incubated with recombinant progranulin (40 nM) with or without leupeptin (100 μM) on ice for 1 h. After washing, cells were shifted to 37 °C and imaged at the indicated time points (seconds). Images were acquired every 12 s for 14 min using live cell confocal microscopy. The background-corrected fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1 and is expressed as a function of time. Error bars represent standard deviation for n = 4 (* P
Figure Legend Snippet: Sortilin levels are stabilized by lysosomal inhibition. Serum-starved PC3/GFP-Sortilin cells were incubated with recombinant progranulin (40 nM) with or without leupeptin (100 μM) on ice for 1 h. After washing, cells were shifted to 37 °C and imaged at the indicated time points (seconds). Images were acquired every 12 s for 14 min using live cell confocal microscopy. The background-corrected fluorescence intensity relative to time 0 was quantified and plotted using the software Zeiss AIM 4.2 SP1 and is expressed as a function of time. Error bars represent standard deviation for n = 4 (* P

Techniques Used: Inhibition, Incubation, Recombinant, Confocal Microscopy, Fluorescence, Software, Standard Deviation

7) Product Images from "Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC"

Article Title: Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC

Journal: The EMBO Journal

doi: 10.1038/emboj.2012.36

Genome-wide screen for starvation-induced autophagy. ( A ) Cellomics images (top) of GFP–LC3-HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom
Figure Legend Snippet: Genome-wide screen for starvation-induced autophagy. ( A ) Cellomics images (top) of GFP–LC3-HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom

Techniques Used: Genome Wide

8) Product Images from "Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2"

Article Title: Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2

Journal: Journal of Virology

doi: 10.1128/JVI.01890-13

Effect of protease inhibitors on virus entry into human lung-derived cell lines. (A) WI-38, MRC-5, and Calu-3 cells were infected with MERS-CoV in the presence of camostat (Camo; 10 μM), EST (10 μM), camostat plus EST (Camo+EST), or leupeptin
Figure Legend Snippet: Effect of protease inhibitors on virus entry into human lung-derived cell lines. (A) WI-38, MRC-5, and Calu-3 cells were infected with MERS-CoV in the presence of camostat (Camo; 10 μM), EST (10 μM), camostat plus EST (Camo+EST), or leupeptin

Techniques Used: Derivative Assay, Infection

9) Product Images from "Metalloproteases regulate T-cell proliferation and effector function via LAG-3"

Article Title: Metalloproteases regulate T-cell proliferation and effector function via LAG-3

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7601520

ADAM10 and ADAM17 cleave LAG-3. ( A ) LAG-3 + -transduced CHO cells were cultured in medium for 1 h±various protease inhibitors (0.5 mM Pefabloc, 100 μM TAPI, 10 μM leupeptin, 10 μM pepstatin A, 10 μM E-64, 25 μM MG-132, 1 μM DAPT or 25 μM LLNL) as indicated in the figure. Supernatants were collected and cells were lysed. Both were immunoprecipitated with the anti-LAG-3 mAb and eluted proteins were separated by SDS–PAGE and blotted with anti-LAG-3.D1 antibody. ( B , C , E , F ) All cells (3A9T cell hybridomas (B), ADAM10 −/+ or ADAM10 −/− MEFs (C), ADAM17 +/+ or ADAM17 ΔZnΔ/Zn fibroblasts (E), CHO and CHO.M1 cells (F)) were transduced with LAG-3 retrovirus. Cells were cultured in medium with DMSO (control), 100 μM TAPI, 1 μM PMA or TAPI and PMA for 0.5 h (E) or 1 h (C). Supernatants and lysates were tested as above. (B–F) The concentration of sLAG-3 in cell culture medium was also assessed by ELISA. LAG-3 concentration was calculated using a standard curve generated with affinity-purified sLAG-3. Data are presented as the mean±s.e. of three separate experiments with P -values shown. ( D ) ADAM10 −/− MEFs were cotransfected with LAG-3 in pMIC (LAG-3) and an IRES-GFP vector pIRES (Vec), a dominant negative bADAM10 cDNA in pIREScg (bADAM10 E−A ) or wild-type bADAM10 in pIREScg. CFP and GFP double-positive cells were sorted and cultured for another 2 days. Cells (1 × 10 6 ) were cultured in 12-well plates. Supernatant and lysate were tested as above. ADAM10 expression was confirmed by blotting whole-cell lysate with anti-ADAM10. The concentration of sLAG-3 in cell culture medium was measured by ELISA. Data are the mean±s.e. of three independent experiments.
Figure Legend Snippet: ADAM10 and ADAM17 cleave LAG-3. ( A ) LAG-3 + -transduced CHO cells were cultured in medium for 1 h±various protease inhibitors (0.5 mM Pefabloc, 100 μM TAPI, 10 μM leupeptin, 10 μM pepstatin A, 10 μM E-64, 25 μM MG-132, 1 μM DAPT or 25 μM LLNL) as indicated in the figure. Supernatants were collected and cells were lysed. Both were immunoprecipitated with the anti-LAG-3 mAb and eluted proteins were separated by SDS–PAGE and blotted with anti-LAG-3.D1 antibody. ( B , C , E , F ) All cells (3A9T cell hybridomas (B), ADAM10 −/+ or ADAM10 −/− MEFs (C), ADAM17 +/+ or ADAM17 ΔZnΔ/Zn fibroblasts (E), CHO and CHO.M1 cells (F)) were transduced with LAG-3 retrovirus. Cells were cultured in medium with DMSO (control), 100 μM TAPI, 1 μM PMA or TAPI and PMA for 0.5 h (E) or 1 h (C). Supernatants and lysates were tested as above. (B–F) The concentration of sLAG-3 in cell culture medium was also assessed by ELISA. LAG-3 concentration was calculated using a standard curve generated with affinity-purified sLAG-3. Data are presented as the mean±s.e. of three separate experiments with P -values shown. ( D ) ADAM10 −/− MEFs were cotransfected with LAG-3 in pMIC (LAG-3) and an IRES-GFP vector pIRES (Vec), a dominant negative bADAM10 cDNA in pIREScg (bADAM10 E−A ) or wild-type bADAM10 in pIREScg. CFP and GFP double-positive cells were sorted and cultured for another 2 days. Cells (1 × 10 6 ) were cultured in 12-well plates. Supernatant and lysate were tested as above. ADAM10 expression was confirmed by blotting whole-cell lysate with anti-ADAM10. The concentration of sLAG-3 in cell culture medium was measured by ELISA. Data are the mean±s.e. of three independent experiments.

Techniques Used: Cell Culture, Immunoprecipitation, SDS Page, Transduction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Generated, Affinity Purification, Plasmid Preparation, Dominant Negative Mutation, Expressing

10) Product Images from "IGFBP-5 Metabolism Is Disrupted in the Rat Medial Meniscal Tear Model of Osteoarthritis"

Article Title: IGFBP-5 Metabolism Is Disrupted in the Rat Medial Meniscal Tear Model of Osteoarthritis

Journal: Cartilage

doi: 10.1177/1947603509359189

Characterization of rat synovial fluid IGFBP-5 protease activity. ( A ) Inhibitor profile. Recombinant IGFBP-5 was incubated with (lanes 3-11) or without (lane 2) synovial fluid lavage from the rat knee 2 weeks after medial meniscal tear (MMT) (pooled samples 1 and 2 from Fig. 1 ). Protease inhibitors were added as follows: lanes 2 and 3 none, lane 4 antipain (250 ug/mL), lane 5 E-64 (60 ug/mL), lane 6 leupeptin (2.5 ug/mL), lane 7 pepstatin (3.5 ug/mL), lane 8 phosphoramidon (1.65 mg/mL), lane 9 Pefabloc (5 mg/mL), lane 10 EDTA-Na 2 (4 mg/mL), and lane 11 aprotinin (10 ug/mL). Samples were processed as in Figure 1 . ( B ) Phenanthroline independence. IGFBP-5 was incubated in the absence (lane 1) or presence (lanes 2 and 3) of synovial fluid lavage. Lane 2: no inhibitor; lane 3: 1 mM 1,10-phenanthroline; lane 4: markers. ( C ) Calcium dependence. IGFBP-5 was incubated with synovial lavage (lanes 2 and 3) in the presence (lane 2) or absence (lane 3) of 4 mM CaCl 2 . Samples were processed as in Figure 1 .
Figure Legend Snippet: Characterization of rat synovial fluid IGFBP-5 protease activity. ( A ) Inhibitor profile. Recombinant IGFBP-5 was incubated with (lanes 3-11) or without (lane 2) synovial fluid lavage from the rat knee 2 weeks after medial meniscal tear (MMT) (pooled samples 1 and 2 from Fig. 1 ). Protease inhibitors were added as follows: lanes 2 and 3 none, lane 4 antipain (250 ug/mL), lane 5 E-64 (60 ug/mL), lane 6 leupeptin (2.5 ug/mL), lane 7 pepstatin (3.5 ug/mL), lane 8 phosphoramidon (1.65 mg/mL), lane 9 Pefabloc (5 mg/mL), lane 10 EDTA-Na 2 (4 mg/mL), and lane 11 aprotinin (10 ug/mL). Samples were processed as in Figure 1 . ( B ) Phenanthroline independence. IGFBP-5 was incubated in the absence (lane 1) or presence (lanes 2 and 3) of synovial fluid lavage. Lane 2: no inhibitor; lane 3: 1 mM 1,10-phenanthroline; lane 4: markers. ( C ) Calcium dependence. IGFBP-5 was incubated with synovial lavage (lanes 2 and 3) in the presence (lane 2) or absence (lane 3) of 4 mM CaCl 2 . Samples were processed as in Figure 1 .

Techniques Used: Activity Assay, Recombinant, Incubation

11) Product Images from "Controlling Proteome Degradation in Daphnia pulex"

Article Title: Controlling Proteome Degradation in Daphnia pulex

Journal: Journal of experimental zoology. Part A, Ecological genetics and physiology

doi: 10.1002/jez.1766

A) Proteolytic activity rates were assessed using an azocasein assay. Absorbance values (+/− standard error) at 440nm were measured for reactions terminated after 10, 30, or 60 minutes. B) Samples were homogenized in RIPA buffer containing the following protease inhibitors: 1) E-64 2) EST(E64-D) 3) Leupeptin 4) Pepstatin A 5) TLCK, HCL 6) TPCK 7) Complete Minitab 8) no inhibitor. Degradation was visually assesed using 1D SDS-PAGE. Samples were either heated at 95°C (H) or not heated (NH) prior to loading to allow degradation to occur during electrophoresis. No protease inhibitor tested was capable of preventing visible degradation and separate samples left at room temperature for six hours had identical degradation patterns to those represented, confirming that degradation did not occur at the homogenization step but only after introduction of denaturing conditions.
Figure Legend Snippet: A) Proteolytic activity rates were assessed using an azocasein assay. Absorbance values (+/− standard error) at 440nm were measured for reactions terminated after 10, 30, or 60 minutes. B) Samples were homogenized in RIPA buffer containing the following protease inhibitors: 1) E-64 2) EST(E64-D) 3) Leupeptin 4) Pepstatin A 5) TLCK, HCL 6) TPCK 7) Complete Minitab 8) no inhibitor. Degradation was visually assesed using 1D SDS-PAGE. Samples were either heated at 95°C (H) or not heated (NH) prior to loading to allow degradation to occur during electrophoresis. No protease inhibitor tested was capable of preventing visible degradation and separate samples left at room temperature for six hours had identical degradation patterns to those represented, confirming that degradation did not occur at the homogenization step but only after introduction of denaturing conditions.

Techniques Used: Activity Assay, Azocasein Assay, SDS Page, Electrophoresis, Protease Inhibitor, Homogenization

12) Product Images from "Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry"

Article Title: Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry

Journal:

doi: 10.1073/pnas.0505577102

Protease-inhibitor sensitivity. ( A ) Leupeptin inhibits S protein-mediated infection. The 293T cells were preincubated with leupeptin and challenged with HIV-luc SARS S (solid line, ♦), VSV-G (dashed line, ▪), or MLV-Ampho (dotted line,
Figure Legend Snippet: Protease-inhibitor sensitivity. ( A ) Leupeptin inhibits S protein-mediated infection. The 293T cells were preincubated with leupeptin and challenged with HIV-luc SARS S (solid line, ♦), VSV-G (dashed line, ▪), or MLV-Ampho (dotted line,

Techniques Used: Protease Inhibitor, Infection

13) Product Images from "Role of Proteases in the Release of Porcine Epidemic Diarrhea Virus from Infected Cells ▿"

Article Title: Role of Proteases in the Release of Porcine Epidemic Diarrhea Virus from Infected Cells ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00464-11

Inhibition of virus release from PEDV-infected Vero/TMPRSS2 cells after treatment with a serine and cysteine protease inhibitor (leupeptin). Vero and Vero/TMPRSS2 cells were infected with PEDV at an MOI of 0.1. After virus adsorption, cells were washed
Figure Legend Snippet: Inhibition of virus release from PEDV-infected Vero/TMPRSS2 cells after treatment with a serine and cysteine protease inhibitor (leupeptin). Vero and Vero/TMPRSS2 cells were infected with PEDV at an MOI of 0.1. After virus adsorption, cells were washed

Techniques Used: Inhibition, Infection, Protease Inhibitor, Adsorption

14) Product Images from "IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome"

Article Title: IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200909067

Phosphorylation of unexpanded polyQ Httex1p and 586aa Htt is associated with its reduced abundance in cell culture. (A) Levels of unexpanded polyQ Httex1p are reduced with overexpression of IKK-β; this effect is inhibited with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4 was cotransfected with myc-actin and with vector or IKK-β into St14A cells. Cells were treated for 16 h with DMSO, 100 nM epoxomicin in DMSO, or 20 mM ammonium chloride/100 µM leupeptin in DMSO. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc to detect myc-actin, and anti-HA to detect Httex1p. (B) IKK-β overexpression reduces levels of unexpanded polyQ Httex1p in the presence of proteasome or lysosome inhibition. Scion software was used to quantitate triplicate levels of 25QP-H4 from the experiment represented in A, normalized to levels of myc-actin transfection control, within each treatment group: control, epoxomicin, or ammonium chloride/leupeptin. (C) Mimicking phosphorylation of unexpanded polyQ Httex1p reduces its abundance in cell culture; this effect is reduced with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4, wt control or QEE, EE, AA, or 3R were cotransfected with myc-actin into St14A cells. Cells and lysates were treated as in A. (D) Levels of phosphorylated unexpanded polyQ 586aa Htt accumulate with inhibition of the proteasome or the lysosome; phosphorylation is reduced with expansion of the polyQ repeat. 15Q or 128Q 586aa Htt constructs were cotransfected into St14A cells with myc-actin and with vector or IKK-β. Cells were treated for 4 h with DMSO or 100 nM epoxomicin in DMSO (to eliminate any possible effect on the lysosome by epoxomicin), or for 16 h with water or 20 mM ammonium chloride/100 µM leupeptin in water. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc, anti–S13-P, and anti-Htt 3B5H10.
Figure Legend Snippet: Phosphorylation of unexpanded polyQ Httex1p and 586aa Htt is associated with its reduced abundance in cell culture. (A) Levels of unexpanded polyQ Httex1p are reduced with overexpression of IKK-β; this effect is inhibited with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4 was cotransfected with myc-actin and with vector or IKK-β into St14A cells. Cells were treated for 16 h with DMSO, 100 nM epoxomicin in DMSO, or 20 mM ammonium chloride/100 µM leupeptin in DMSO. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc to detect myc-actin, and anti-HA to detect Httex1p. (B) IKK-β overexpression reduces levels of unexpanded polyQ Httex1p in the presence of proteasome or lysosome inhibition. Scion software was used to quantitate triplicate levels of 25QP-H4 from the experiment represented in A, normalized to levels of myc-actin transfection control, within each treatment group: control, epoxomicin, or ammonium chloride/leupeptin. (C) Mimicking phosphorylation of unexpanded polyQ Httex1p reduces its abundance in cell culture; this effect is reduced with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4, wt control or QEE, EE, AA, or 3R were cotransfected with myc-actin into St14A cells. Cells and lysates were treated as in A. (D) Levels of phosphorylated unexpanded polyQ 586aa Htt accumulate with inhibition of the proteasome or the lysosome; phosphorylation is reduced with expansion of the polyQ repeat. 15Q or 128Q 586aa Htt constructs were cotransfected into St14A cells with myc-actin and with vector or IKK-β. Cells were treated for 4 h with DMSO or 100 nM epoxomicin in DMSO (to eliminate any possible effect on the lysosome by epoxomicin), or for 16 h with water or 20 mM ammonium chloride/100 µM leupeptin in water. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc, anti–S13-P, and anti-Htt 3B5H10.

Techniques Used: Cell Culture, Over Expression, Plasmid Preparation, Western Blot, Inhibition, Software, Transfection, Construct

15) Product Images from "Oxidative Stress Enhances Neurodegeneration Markers Induced by Herpes Simplex Virus Type 1 Infection in Human Neuroblastoma Cells"

Article Title: Oxidative Stress Enhances Neurodegeneration Markers Induced by Herpes Simplex Virus Type 1 Infection in Human Neuroblastoma Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0075842

Effects of oxidative stress on autophagy. A) SK-LC3 cells were simultaneously treated with X-XOD and infected with HSV-1 at a moi of 10 for 18 h and the cellular distribution of GFP-LC3 assessed by fluorescence microscopy. Scale bar: 10 µm. B) Analysis of GFP-LC3 levels by Western blotting with an anti-GFP antibody in SK-LC3 cells simultaneously treated with X-XOD and infected with HSV-1 (moi of 10 for 18 h). The effects of the lysosomal inhibitors leupeptin and ammonium chloride are shown. Blots are representative of four independent experiments; a tubulin blot was performed as a loading control. The ratio of LC3-II to tubulin is shown below the blots. C) The graph represents the fold increase in GFP-LC3-II levels, normalized by tubulin, in leupeptin/NH 4 Cl-treated cells compared to non-treated cells in all conditions assayed in ( B ).
Figure Legend Snippet: Effects of oxidative stress on autophagy. A) SK-LC3 cells were simultaneously treated with X-XOD and infected with HSV-1 at a moi of 10 for 18 h and the cellular distribution of GFP-LC3 assessed by fluorescence microscopy. Scale bar: 10 µm. B) Analysis of GFP-LC3 levels by Western blotting with an anti-GFP antibody in SK-LC3 cells simultaneously treated with X-XOD and infected with HSV-1 (moi of 10 for 18 h). The effects of the lysosomal inhibitors leupeptin and ammonium chloride are shown. Blots are representative of four independent experiments; a tubulin blot was performed as a loading control. The ratio of LC3-II to tubulin is shown below the blots. C) The graph represents the fold increase in GFP-LC3-II levels, normalized by tubulin, in leupeptin/NH 4 Cl-treated cells compared to non-treated cells in all conditions assayed in ( B ).

Techniques Used: Infection, Fluorescence, Microscopy, Western Blot

16) Product Images from "Most rotavirus strains require the cation-independent mannose-6-phosphate receptor, sortilin-1, and cathepsins to enter cells"

Article Title: Most rotavirus strains require the cation-independent mannose-6-phosphate receptor, sortilin-1, and cathepsins to enter cells

Journal: Virus Research

doi: 10.1016/j.virusres.2017.12.002

Effect of cathepsin inhibitors on the infectivity of rotavirus in MA104 cells. MA104 cells were treated or mock treated with (A) leupeptin (25 μM), (B) the cathepsin B inhibitor CA-074 (5 μM) or, (C) the cathepsin L inhibitor Z-FF-FMK (5 μM) for 1 h at 37 °C, and then infected with the indicated viruses at an MOI of 0.02. Cells infected with the indicated rotavirus strain or reovirus type 1 (ReoT1L) were fixed and immunostained at 14 or 16 hpi, respectively, as described in Section 2.5 . Data are expressed as the percent infectivity of each virus compared to the infectivity obtained in mock-treated cells. The arithmetic means ± SEM of at least two independent experiments performed in duplicate are shown. The asterisks indicate significant differences between the infectivity of each virus in inhibitor-treated cells with respect to mock-treated cells. *, P
Figure Legend Snippet: Effect of cathepsin inhibitors on the infectivity of rotavirus in MA104 cells. MA104 cells were treated or mock treated with (A) leupeptin (25 μM), (B) the cathepsin B inhibitor CA-074 (5 μM) or, (C) the cathepsin L inhibitor Z-FF-FMK (5 μM) for 1 h at 37 °C, and then infected with the indicated viruses at an MOI of 0.02. Cells infected with the indicated rotavirus strain or reovirus type 1 (ReoT1L) were fixed and immunostained at 14 or 16 hpi, respectively, as described in Section 2.5 . Data are expressed as the percent infectivity of each virus compared to the infectivity obtained in mock-treated cells. The arithmetic means ± SEM of at least two independent experiments performed in duplicate are shown. The asterisks indicate significant differences between the infectivity of each virus in inhibitor-treated cells with respect to mock-treated cells. *, P

Techniques Used: Infection

Effect of protease inhibitors on the infectivity of different rotavirus strains in Caco-2 cells. Caco-2 cells were treated or mock-treated with (A) leupeptin, (B) the cathepsin B inhibitor CA-074 or, (C) the cathepsin L inhibitor Z-FF-FMK, and infected with the indicated viruses at an MOI of 0.02. At 14 or 16 hpi (for assays with rotaviruses or reovirus, respectively) cells were fixed and immunostained as described in Section 2.5 . Data are expressed as the percent infectivity of each virus compared to the infectivity obtained in mock-treated cells. The arithmetic means ± SEM of at least two independent experiments performed in duplicate are shown. The asterisks indicate significant differences between the infectivity of each virus in inhibitor-treated cells with respect to mock-treated cells. **, P
Figure Legend Snippet: Effect of protease inhibitors on the infectivity of different rotavirus strains in Caco-2 cells. Caco-2 cells were treated or mock-treated with (A) leupeptin, (B) the cathepsin B inhibitor CA-074 or, (C) the cathepsin L inhibitor Z-FF-FMK, and infected with the indicated viruses at an MOI of 0.02. At 14 or 16 hpi (for assays with rotaviruses or reovirus, respectively) cells were fixed and immunostained as described in Section 2.5 . Data are expressed as the percent infectivity of each virus compared to the infectivity obtained in mock-treated cells. The arithmetic means ± SEM of at least two independent experiments performed in duplicate are shown. The asterisks indicate significant differences between the infectivity of each virus in inhibitor-treated cells with respect to mock-treated cells. **, P

Techniques Used: Infection

17) Product Images from "PI3P signaling regulates receptor sorting but not transport in the endosomal pathway"

Article Title: PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200303018

GFP-2xFYVE does not inhibit bulk transport to late endosomes in vivo. (A) As in Fig. 1 A, a pulse of rhodamine dextran was endocytosed with or without chase in cells expressing a double FYVE domain associated to GFP (GFP-2xFYVE). Cells were analyzed as described in the legend to Fig. 1 A. (B) Cells expressing or not expressing GFP-2xFYVE were incubated overnight with 40 μg/ml mouse IgGs with or without 50 μg/ml leupeptin. Endocytosed IgGs were revealed using anti-mouse antibodies and cells were analyzed by double channel fluorescence microscopy. The total number of labeled vesicles (containing IgGs in red) was counted for each condition in control cells or cells expressing GFP-2xFYVE. Vesicles in ≥20 cells were counted for each condition in three different experiments. Numbers were similar whether GFP-2xFYVE was present or not, and are expressed as a percentage of the total number of vesicles in leupeptin-treated cells, to facilitate direct comparison. Bar, 2.5 μm.
Figure Legend Snippet: GFP-2xFYVE does not inhibit bulk transport to late endosomes in vivo. (A) As in Fig. 1 A, a pulse of rhodamine dextran was endocytosed with or without chase in cells expressing a double FYVE domain associated to GFP (GFP-2xFYVE). Cells were analyzed as described in the legend to Fig. 1 A. (B) Cells expressing or not expressing GFP-2xFYVE were incubated overnight with 40 μg/ml mouse IgGs with or without 50 μg/ml leupeptin. Endocytosed IgGs were revealed using anti-mouse antibodies and cells were analyzed by double channel fluorescence microscopy. The total number of labeled vesicles (containing IgGs in red) was counted for each condition in control cells or cells expressing GFP-2xFYVE. Vesicles in ≥20 cells were counted for each condition in three different experiments. Numbers were similar whether GFP-2xFYVE was present or not, and are expressed as a percentage of the total number of vesicles in leupeptin-treated cells, to facilitate direct comparison. Bar, 2.5 μm.

Techniques Used: In Vivo, Expressing, Incubation, Fluorescence, Microscopy, Labeling

PI3P signaling regulates EGFR sorting in early endosomes. (A) Cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A) were incubated for 10 min at 37°C with rhodamine-dextran, followed by a 90-min chase with 100 nM wortmannin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (B) Cells transfected with GFP-2xFYVE were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C followed (chase) or not (pulse) by a 90-min chase in the presence of leupeptin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (C) Cells expressing GFP-2xFYVE (FYVE), or treated (wort) or not treated (control: ctrl) with wortmannin were labeled with endocytosed EGF-biotin/streptavidin-phycoerythrin (as in B) or dextran (as in A), except that the chase time period was 45 min, and then processed for immunofluorescence using antibodies against the early endosomal (EE) marker EEA1 or the late endosomal (LE) marker LBPA, as indicated. For each condition, the total number of vesicles labeled with EGF or dextran was counted from ≥15 cells in three different experiments (expressed as 100%), as well as the percentage of these vesicles that also contained the EE (EEA1 in control cells; FYVE in FYVE-expressing cells) or the LE endosomal marker LBPA (except with wortmannin, since the drug releases EEA1). (D) After EGF pretreatment (as in Fig. 1 A), cells overexpressing EGFR-GFP were incubated for 10 min at 37°C followed (chase) or not (pulse) by a 35-min chase with wortmannin (wort) and processed for immunofluorescence. (E) The number of vesicles in D that contained both GFP-EGFR and TfR was counted, and is expressed as a percentage of the total number of GFP-EGFR–positive vesicles (C). Bars: (A and B) 2.5 μm; (D) 5 μm.
Figure Legend Snippet: PI3P signaling regulates EGFR sorting in early endosomes. (A) Cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A) were incubated for 10 min at 37°C with rhodamine-dextran, followed by a 90-min chase with 100 nM wortmannin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (B) Cells transfected with GFP-2xFYVE were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C followed (chase) or not (pulse) by a 90-min chase in the presence of leupeptin, labeled with antibodies against the indicated antigens and analyzed by triple channel fluorescence. (C) Cells expressing GFP-2xFYVE (FYVE), or treated (wort) or not treated (control: ctrl) with wortmannin were labeled with endocytosed EGF-biotin/streptavidin-phycoerythrin (as in B) or dextran (as in A), except that the chase time period was 45 min, and then processed for immunofluorescence using antibodies against the early endosomal (EE) marker EEA1 or the late endosomal (LE) marker LBPA, as indicated. For each condition, the total number of vesicles labeled with EGF or dextran was counted from ≥15 cells in three different experiments (expressed as 100%), as well as the percentage of these vesicles that also contained the EE (EEA1 in control cells; FYVE in FYVE-expressing cells) or the LE endosomal marker LBPA (except with wortmannin, since the drug releases EEA1). (D) After EGF pretreatment (as in Fig. 1 A), cells overexpressing EGFR-GFP were incubated for 10 min at 37°C followed (chase) or not (pulse) by a 35-min chase with wortmannin (wort) and processed for immunofluorescence. (E) The number of vesicles in D that contained both GFP-EGFR and TfR was counted, and is expressed as a percentage of the total number of GFP-EGFR–positive vesicles (C). Bars: (A and B) 2.5 μm; (D) 5 μm.

Techniques Used: Expressing, Incubation, Labeling, Fluorescence, Transfection, Immunofluorescence, Marker

Transport from early to late endosomes after PI3 kinase inhibition. (A) BHK cells expressing EGFR-GFP were preincubated with EGF for 1 h at 4°C and washed; this pretreatment was always used for EGF or EGFR endocytosis. Cells were incubated for 10 min at 37°C with rhodamine-dextran (pulse), labeled with antibodies against the indicated antigens, and analyzed by triple channel fluorescence microscopy. (B) BHK cells overexpressing EGFR-GFP were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C, followed by a 90-min chase in the presence of 0.5 mg/ml leupeptin and analyzed as in described for A; high magnification views of the indicated areas are shown below the micrographs. (C) A pulse of rhodamine dextran was endocytosed as described for A and then chased for 45 min; cells were analyzed as described for A. (D) A pulse of rhodamine dextran was endocytosed as described for A into BHK cells with or without chase (as in C); 100 nM wortmannin (Wort) was present during both the pulse and the chase. Cells were analyzed as described for A. Bars: (A–C) 5 μm; (D) 2.5 μm.
Figure Legend Snippet: Transport from early to late endosomes after PI3 kinase inhibition. (A) BHK cells expressing EGFR-GFP were preincubated with EGF for 1 h at 4°C and washed; this pretreatment was always used for EGF or EGFR endocytosis. Cells were incubated for 10 min at 37°C with rhodamine-dextran (pulse), labeled with antibodies against the indicated antigens, and analyzed by triple channel fluorescence microscopy. (B) BHK cells overexpressing EGFR-GFP were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C, followed by a 90-min chase in the presence of 0.5 mg/ml leupeptin and analyzed as in described for A; high magnification views of the indicated areas are shown below the micrographs. (C) A pulse of rhodamine dextran was endocytosed as described for A and then chased for 45 min; cells were analyzed as described for A. (D) A pulse of rhodamine dextran was endocytosed as described for A into BHK cells with or without chase (as in C); 100 nM wortmannin (Wort) was present during both the pulse and the chase. Cells were analyzed as described for A. Bars: (A–C) 5 μm; (D) 2.5 μm.

Techniques Used: Inhibition, Expressing, Incubation, Labeling, Fluorescence, Microscopy

18) Product Images from "Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity"

Article Title: Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity

Journal: Virology Journal

doi: 10.1186/1743-422X-2-63

Effect of inhibitors on in vitro processing . Various concentrations of protease inhibitors were added to the in vitro processing assay for 6 hr at 29°C. The first lane is P25K expressed alone with no extract added. The second lane is P25K mixed with cellular extracts and the third lane is P25K mixed with I7L enzyme extracts. Each of the remaining lanes has P25K mixed with I7L enzyme extracts plus indicated inhibitor. Ethylenediaminetetraacetic acid (EDTA) was used at 1 mM. Pepstatin A, Pep, was used at 10 μM. Phenlymethanesulfonyl fluoride (PMSF) was used at 1 mM. N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide trans -Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) and a related product EST, were both used at 10 μM and 100 μM concentrations. Iodoacetic acid (IA) was used at 1 mM. Leupeptin (Leu) was used at 1 mM, and N-ethlymaleimide (NEM) was used at 2.5 mM. The concentrations of TTP-6171, TTP-1021, and TTP-0961 are indicated. The table indicates the concentration of inhibitor used and whether cleavage activity was observed.
Figure Legend Snippet: Effect of inhibitors on in vitro processing . Various concentrations of protease inhibitors were added to the in vitro processing assay for 6 hr at 29°C. The first lane is P25K expressed alone with no extract added. The second lane is P25K mixed with cellular extracts and the third lane is P25K mixed with I7L enzyme extracts. Each of the remaining lanes has P25K mixed with I7L enzyme extracts plus indicated inhibitor. Ethylenediaminetetraacetic acid (EDTA) was used at 1 mM. Pepstatin A, Pep, was used at 10 μM. Phenlymethanesulfonyl fluoride (PMSF) was used at 1 mM. N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide trans -Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) and a related product EST, were both used at 10 μM and 100 μM concentrations. Iodoacetic acid (IA) was used at 1 mM. Leupeptin (Leu) was used at 1 mM, and N-ethlymaleimide (NEM) was used at 2.5 mM. The concentrations of TTP-6171, TTP-1021, and TTP-0961 are indicated. The table indicates the concentration of inhibitor used and whether cleavage activity was observed.

Techniques Used: In Vitro, IA, Concentration Assay, Activity Assay

19) Product Images from "Role of Proteases in the Release of Porcine Epidemic Diarrhea Virus from Infected Cells ▿"

Article Title: Role of Proteases in the Release of Porcine Epidemic Diarrhea Virus from Infected Cells ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00464-11

Inhibition of virus release from PEDV-infected Vero/TMPRSS2 cells after treatment with a serine and cysteine protease inhibitor (leupeptin). Vero and Vero/TMPRSS2 cells were infected with PEDV at an MOI of 0.1. After virus adsorption, cells were washed
Figure Legend Snippet: Inhibition of virus release from PEDV-infected Vero/TMPRSS2 cells after treatment with a serine and cysteine protease inhibitor (leupeptin). Vero and Vero/TMPRSS2 cells were infected with PEDV at an MOI of 0.1. After virus adsorption, cells were washed

Techniques Used: Inhibition, Infection, Protease Inhibitor, Adsorption

20) Product Images from "Gilenya (FTY720) inhibits acid sphingomyelinase by a mechanism similar to tricyclic antidepressants"

Article Title: Gilenya (FTY720) inhibits acid sphingomyelinase by a mechanism similar to tricyclic antidepressants

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2010.11.115

FTY720 (but not S1P) inhibits ASMase in cultured cells, and leupeptin (Leu) can reverse the inhibition FTY720 and S1P were added at 10μM and leupeptin at 5μM as described in the text. Results were based on multiple assays run in triplicate and are expressed as percent of control activity.
Figure Legend Snippet: FTY720 (but not S1P) inhibits ASMase in cultured cells, and leupeptin (Leu) can reverse the inhibition FTY720 and S1P were added at 10μM and leupeptin at 5μM as described in the text. Results were based on multiple assays run in triplicate and are expressed as percent of control activity.

Techniques Used: Cell Culture, Inhibition, Activity Assay

FTY720 does not affect the message levels for ASMase even when inhibiting activity > 90% Panel A: RT-PCR of human LA-N-5 cells. Lane 1, control; lane 2, 10μM FTY720 treatment; Lane 3, 5μM Leupeptin pre-incubation for 1h followed by 10μM FTY720 treatment. β-Actin is the control for sample application. Panel B: Western blot of human LA-N-5 cells. Lane1, control; Lane2, 10μM FTY720 treatment; Lane 3, 5μM Leupeptin pre-incubation for 1h then 10μM FTY720 treatment for 24h. β-Actin is the control for sample application.
Figure Legend Snippet: FTY720 does not affect the message levels for ASMase even when inhibiting activity > 90% Panel A: RT-PCR of human LA-N-5 cells. Lane 1, control; lane 2, 10μM FTY720 treatment; Lane 3, 5μM Leupeptin pre-incubation for 1h followed by 10μM FTY720 treatment. β-Actin is the control for sample application. Panel B: Western blot of human LA-N-5 cells. Lane1, control; Lane2, 10μM FTY720 treatment; Lane 3, 5μM Leupeptin pre-incubation for 1h then 10μM FTY720 treatment for 24h. β-Actin is the control for sample application.

Techniques Used: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Western Blot

21) Product Images from "TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes"

Article Title: TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201111079

TBC1D14 binds Rab11, and inactive Rab11 inhibits autophagy. (A) Yeast two-hybrid screen to identify Rab-binding partners of TBC1D14. Yeast were transfected with the active (QA) Rab constructs (prey) and TBC1D14R472A (bait) and selected on plates lacking leucine and tryptophan (−L/−W) and medium lacking leucine, tryptophan, histidine, and adenine (QDO). Five colonies for each Rab were streaked on selective media. (B) HEK293A cells were transfected with GFP-tagged Rab11 constructs, 11aWT, 11aQ70A, 11aN124I, and 11bN124I and, 24 h later, fed or starved for 2 h. Western blot of a representative experiment with anti-GFP, anti-LC3, and antiactin and quantification of LC3-II/actin. n ≥ 3; one-way ANOVA followed by Bonferroni posttest analysis comparing to the vector control are starved with leupeptin (leu) versus Rab11aN124I starved with leupeptin: *, P
Figure Legend Snippet: TBC1D14 binds Rab11, and inactive Rab11 inhibits autophagy. (A) Yeast two-hybrid screen to identify Rab-binding partners of TBC1D14. Yeast were transfected with the active (QA) Rab constructs (prey) and TBC1D14R472A (bait) and selected on plates lacking leucine and tryptophan (−L/−W) and medium lacking leucine, tryptophan, histidine, and adenine (QDO). Five colonies for each Rab were streaked on selective media. (B) HEK293A cells were transfected with GFP-tagged Rab11 constructs, 11aWT, 11aQ70A, 11aN124I, and 11bN124I and, 24 h later, fed or starved for 2 h. Western blot of a representative experiment with anti-GFP, anti-LC3, and antiactin and quantification of LC3-II/actin. n ≥ 3; one-way ANOVA followed by Bonferroni posttest analysis comparing to the vector control are starved with leupeptin (leu) versus Rab11aN124I starved with leupeptin: *, P

Techniques Used: Two Hybrid Screening, Binding Assay, Transfection, Construct, Western Blot, Plasmid Preparation

A screen of 38 human RabGAPs reveals that 11 inhibit autophagy. (A) An overexpression screen for RabGAPs, which are autophagy inhibitors. 38 myc-TBC domain–containing proteins were expressed in 2GL9 cells for 24 h, and then, cells were fed or starved in EBSS with leupeptin (leu) for 2 h. GFP–LC3-II was quantified by the Odyssey system with antibodies against LC3, actin, and myc. Black bars show levels of GFP–LC3-II after starvation normalized to the renilla vector control. Gray bars show the effect of overexpression of eight GAP activity–deficient RA mutants. The data shown are from single representative experiments. All myc-TBC transfections were repeated ( n ≥ 2) and, if inhibitory or if performed with the RA mutant, repeated three or more times ( n ≥ 3). ULK1ΔC, an inhibitory C-terminal truncation mutant ( Chan et al., 2009 ) was included in each experiment. No catalytic arginine could be identified in TBC1D4, TBC1D9, or TBC1D7. Horizontal lines illustrate the value of the vector control (set to 1) and 60% value of the control. (B) Representative Western blots for myc, GFP-LC3, and actin from the 11 myc-TBC proteins in a screen that inhibited autophagy by 40% or more. The asterisk indicates a nonspecific band. MM, molecular mass; WB, Western blot.
Figure Legend Snippet: A screen of 38 human RabGAPs reveals that 11 inhibit autophagy. (A) An overexpression screen for RabGAPs, which are autophagy inhibitors. 38 myc-TBC domain–containing proteins were expressed in 2GL9 cells for 24 h, and then, cells were fed or starved in EBSS with leupeptin (leu) for 2 h. GFP–LC3-II was quantified by the Odyssey system with antibodies against LC3, actin, and myc. Black bars show levels of GFP–LC3-II after starvation normalized to the renilla vector control. Gray bars show the effect of overexpression of eight GAP activity–deficient RA mutants. The data shown are from single representative experiments. All myc-TBC transfections were repeated ( n ≥ 2) and, if inhibitory or if performed with the RA mutant, repeated three or more times ( n ≥ 3). ULK1ΔC, an inhibitory C-terminal truncation mutant ( Chan et al., 2009 ) was included in each experiment. No catalytic arginine could be identified in TBC1D4, TBC1D9, or TBC1D7. Horizontal lines illustrate the value of the vector control (set to 1) and 60% value of the control. (B) Representative Western blots for myc, GFP-LC3, and actin from the 11 myc-TBC proteins in a screen that inhibited autophagy by 40% or more. The asterisk indicates a nonspecific band. MM, molecular mass; WB, Western blot.

Techniques Used: Over Expression, Plasmid Preparation, Activity Assay, Transfection, Mutagenesis, Western Blot

22) Product Images from "Rotaviruses Reach Late Endosomes and Require the Cation-Dependent Mannose-6-Phosphate Receptor and the Activity of Cathepsin Proteases To Enter the Cell"

Article Title: Rotaviruses Reach Late Endosomes and Require the Cation-Dependent Mannose-6-Phosphate Receptor and the Activity of Cathepsin Proteases To Enter the Cell

Journal: Journal of Virology

doi: 10.1128/JVI.03457-13

Role of cathepsins in RV infectivity. MA104 cells were treated or mock treated with leupeptin (A), the cathepsin B (CTS-B) inhibitor CA-074 (B), or the cathepsin L (CTS-L) inhibitor Z-FF-FMK (C) and infected with the indicated viruses at an MOI of 0.02.
Figure Legend Snippet: Role of cathepsins in RV infectivity. MA104 cells were treated or mock treated with leupeptin (A), the cathepsin B (CTS-B) inhibitor CA-074 (B), or the cathepsin L (CTS-L) inhibitor Z-FF-FMK (C) and infected with the indicated viruses at an MOI of 0.02.

Techniques Used: Infection

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Incubation:

Article Title: Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor
Article Snippet: .. Treatment with lysosomal inhibitors Mock- and siRNA-treated cells were incubated for 3 h at 37°C in complete DME containing 1 mg/ml leupeptin (Roche) and 5 μg/ml E64 (Sigma-Aldrich) plus or minus 10 mM methionine methyl ester (Sigma-Aldrich). .. Cells were scraped, lysed, and analyzed by SDS-PAGE (6% acrylamide or 4–20% acrylamide gradient) and immunoblotting.

Article Title: Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC
Article Snippet: .. The plates were washed twice with PBS and incubated in amino acid-free ES (Earle's Buffered Saline Solution) supplemented with leupeptin (0.25 mg/ml; Roche) (EL) for 2 h at 37°C. .. Cells were fixed with 8% formaldehyde (Sigma) in PBS at 4°C (final concentration 4% formaldehyde) and stained with DAPI (1 μg/ml) for 15 min and stored at 4°C in PBS.

other:

Article Title: Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2
Article Snippet: In addition, the following inhibitors were used: EST [(23,25)- trans -epoxysuccinyl- l -leucylamindo-3-methylbutane ethyl ester; catalog no. 330005; Calbiochem], bafilomycin A1 (catalog no. B1793; Sigma), cathepsin L inhibitor III (catalog no. 219427; Calbiochem), cathepsin B inhibitor CA-074 (catalog no. C5857; Sigma), cathepsin K inhibitor II (Z-L-NHNHCONHNH-LF-Boc; catalog no. 219379; Calbiochem), cathepsin S inhibitor (Z-FL-Cocho; catalog no. 219393; Calbiochem), leupeptin (product no. 11017101001; Roche), and camostat mesylate (catalog no. 3193; Tocris Bioscience).

Thin Layer Chromatography:

Article Title: Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling
Article Snippet: .. Phenylmethylsulfonylfluoride, leupeptin, and aprotinin were obtained from Roche Molecular Biochemicals; paraformaldehyde and anti-actin antibody from Sigma (St. Louis, MO); [3 H]palmitic acid and [32 P]orthophosphate from Dupont NEN (Boston, MA); [3 H]myristic acid and the chemiluminescence kit (ECL system) from Amersham International (Buckinghamshire, U.K.); Silica Gel 60 TLC plates from Merck (Darmstadt, Germany); immobilized protein A and rhodamine-conjugated anti-mouse antibody from Pierce (Rockford, IL); DMEM and LipofectAmine from Gibco-BRL (Grand Island, NY); fetal bovine serum from PAA Laboratories, Inc. (Parker Ford, PA); and horseradish peroxidase–conjugated goat anti-rabbit IgG or anti-mouse IgG, IgM, and IgA from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD). .. The antibody against the C-terminal region of PLD1 was made and purified as described previously ( ).

Immunoprecipitation:

Article Title: The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin
Article Snippet: .. Lysates from several plates were pooled, and 2 mg of proteins were immunoprecipitated in HNTG buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.2 mM sodium orthovanadate, protease inhibitor mix [Roche]) supplemented with 20 μM of the proteasome inhibitor MG132 (Calbiochem) and 100 μM of leupeptin as lysosomal inhibitors (Roche) to accumulate the ubiquitinated species. .. Filters were immunoblotted with anti-HA monoclonal antibodies (P4D1, Covance) to detect ubiquitinated proteins.

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    Roche leupeptin
    Schematic representation of aSMase processing. A , pro-aSMase (72–75 kDa) arises from prepro-aSMase (75 kDa) within the Golgi and is either targeted to the lysosome via mannose 6-phosphorylation ( M6P ), or is released extracellularly through the default Golgi secretory pathway giving rise to S-SMase (75–80 kDa). Upon entry to the endolysosomal compartment pro-aSMase is cleaved by “protease X” giving rise to the 65-kDa aSMase, which upon encountering Zn 2+ in the lysosome becomes L-SMase. L-SMase (65 kDa) is in turn degraded by “protease Y” to the 52-kDa inactive L-SMase. The action of protease Y is promoted by desipramine, and inhibited by <t>leupeptin.</t> B , both pro-aSMase and S-SMase have an intact C terminus, require exogenous Zn 2+ for activity, and are insensitive to desipramine. Mature L-SMase has lost a portion of its C terminus, which may promote coordination of lysosomal Zn 2+ , thereby rendering the 65-kDa aSMase, Zn 2+ -independent, and is sensitive to desipramine.
    Leupeptin, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic representation of aSMase processing. A , pro-aSMase (72–75 kDa) arises from prepro-aSMase (75 kDa) within the Golgi and is either targeted to the lysosome via mannose 6-phosphorylation ( M6P ), or is released extracellularly through the default Golgi secretory pathway giving rise to S-SMase (75–80 kDa). Upon entry to the endolysosomal compartment pro-aSMase is cleaved by “protease X” giving rise to the 65-kDa aSMase, which upon encountering Zn 2+ in the lysosome becomes L-SMase. L-SMase (65 kDa) is in turn degraded by “protease Y” to the 52-kDa inactive L-SMase. The action of protease Y is promoted by desipramine, and inhibited by leupeptin. B , both pro-aSMase and S-SMase have an intact C terminus, require exogenous Zn 2+ for activity, and are insensitive to desipramine. Mature L-SMase has lost a portion of its C terminus, which may promote coordination of lysosomal Zn 2+ , thereby rendering the 65-kDa aSMase, Zn 2+ -independent, and is sensitive to desipramine.

    Journal: The Journal of Biological Chemistry

    Article Title: A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

    doi: 10.1074/jbc.M110.155234

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

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

    Techniques: Activity Assay

    Complex formation of PLD1 with caveolin-1 and with the EGFR in the CEM. COS-7 cells transfected with WT or C240S/C241S mutant of PLD1 were incubated in the presence or absence of 100 nM of EGF for 0.5 min. The CEM fraction was prepared, and the proteins were solubilized in an extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Nonidet P-40, 0.4% deoxycholate, 60 mM β-octylglucopyranoside, 0.5 mM phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, and 5 μg/ml aprotinin). The sample was centrifuged at 150,000 × g for 1 h, and the solubilized supernatant was incubated with anti-PLD antibody precoupled to protein A agarose resin. The immune complexes were subjected to 6–16% gradient SDS-PAGE followed by immunoblot analysis with anti-PLD, anti-EGFR, or anti-caveolin-1 antibody. The blots shown are representative of three separate results. I.P., immunoprecipitate.

    Journal: Molecular Biology of the Cell

    Article Title: Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

    doi: 10.1091/mbc.E02-02-0100

    Figure Lengend Snippet: Complex formation of PLD1 with caveolin-1 and with the EGFR in the CEM. COS-7 cells transfected with WT or C240S/C241S mutant of PLD1 were incubated in the presence or absence of 100 nM of EGF for 0.5 min. The CEM fraction was prepared, and the proteins were solubilized in an extraction buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Nonidet P-40, 0.4% deoxycholate, 60 mM β-octylglucopyranoside, 0.5 mM phenylmethylsulfonylfluoride, 1 μg/ml leupeptin, and 5 μg/ml aprotinin). The sample was centrifuged at 150,000 × g for 1 h, and the solubilized supernatant was incubated with anti-PLD antibody precoupled to protein A agarose resin. The immune complexes were subjected to 6–16% gradient SDS-PAGE followed by immunoblot analysis with anti-PLD, anti-EGFR, or anti-caveolin-1 antibody. The blots shown are representative of three separate results. I.P., immunoprecipitate.

    Article Snippet: Phenylmethylsulfonylfluoride, leupeptin, and aprotinin were obtained from Roche Molecular Biochemicals; paraformaldehyde and anti-actin antibody from Sigma (St. Louis, MO); [3 H]palmitic acid and [32 P]orthophosphate from Dupont NEN (Boston, MA); [3 H]myristic acid and the chemiluminescence kit (ECL system) from Amersham International (Buckinghamshire, U.K.); Silica Gel 60 TLC plates from Merck (Darmstadt, Germany); immobilized protein A and rhodamine-conjugated anti-mouse antibody from Pierce (Rockford, IL); DMEM and LipofectAmine from Gibco-BRL (Grand Island, NY); fetal bovine serum from PAA Laboratories, Inc. (Parker Ford, PA); and horseradish peroxidase–conjugated goat anti-rabbit IgG or anti-mouse IgG, IgM, and IgA from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD).

    Techniques: Transfection, Mutagenesis, Incubation, SDS Page

    Tetraspanin effects on MT1-MMP-dependent fibronectin degradation. MCF-7-VC and MCF-7-MT1 cells were treated with siRNAs, plated 4 d later on fibronectin (FN)-coated slides, and then incubated overnight in serum-free media with protease inhibitors (50 μg/ml aprotinin, 2 μM leupeptin, 20 μM E64, and 20 μM pepstatin A) to prevent non-MMP proteolysis of the FN. The following day, cells were washed, fixed, and FN was detected using an anti-FN antibody followed by Alexa 488-conjugated anti-mouse secondary antibody (green). The position of the cells was determined by staining the cytoskeleton with Alexa Fluor 546-phalloidin (red) and the nuclei with DAPI (blue). Representative fluorescent images (merge green/red/blue or green only) are shown. Bar, 20 μm. For the bar graph, 100% fibronectin degradation is defined as 0 pixel density within defined proteolyzed areas, whereas 0% degradation is pixel density in the unperturbed fibronectin layer. Results are mean of percentage of fibronectin degradation ± SD (n = 3; *p

    Journal: Molecular Biology of the Cell

    Article Title: Tetraspanin Proteins Regulate Membrane Type-1 Matrix Metalloproteinase-dependent Pericellular Proteolysis

    doi: 10.1091/mbc.E08-11-1149

    Figure Lengend Snippet: Tetraspanin effects on MT1-MMP-dependent fibronectin degradation. MCF-7-VC and MCF-7-MT1 cells were treated with siRNAs, plated 4 d later on fibronectin (FN)-coated slides, and then incubated overnight in serum-free media with protease inhibitors (50 μg/ml aprotinin, 2 μM leupeptin, 20 μM E64, and 20 μM pepstatin A) to prevent non-MMP proteolysis of the FN. The following day, cells were washed, fixed, and FN was detected using an anti-FN antibody followed by Alexa 488-conjugated anti-mouse secondary antibody (green). The position of the cells was determined by staining the cytoskeleton with Alexa Fluor 546-phalloidin (red) and the nuclei with DAPI (blue). Representative fluorescent images (merge green/red/blue or green only) are shown. Bar, 20 μm. For the bar graph, 100% fibronectin degradation is defined as 0 pixel density within defined proteolyzed areas, whereas 0% degradation is pixel density in the unperturbed fibronectin layer. Results are mean of percentage of fibronectin degradation ± SD (n = 3; *p

    Article Snippet: Protein G-agarose beads, aprotinin, and leupeptin (serine and thiol protease inhibitor) were from Roche Diagnostics.

    Techniques: Incubation, Staining

    Both CLR-mediated and CD4-mediated endocytosis of Env result in similar kinetics of antigen processing Cells were incubated with 10µg/mL FITC-labeled Env or HA for 90 min at 4°C or 37°C, then washed twice and cultured at 4°C or 37°C for up to 24h. In some cases, MDCs were pre-incubated and the experiment carried out in the presence of 1mM leupeptin (Leu) to inhibit proteolytic degradation. Samples were then stained with DC markers for analysis by flow cytometry and gated on HLA-DR + /CD11c + (MDCs), CD123 + (PDCs), CD1a + (LCs) or HLA-DR + (DDCs). A) Representative MDC donor showing the percentage of positive cells. B) The MFI for each timepoint was normalised to the 0 min 37°C control for each Ag and the data shown here are the mean ± SD for for 3–5 donors for Env and 1 donor for HA. C) Alternatively, MDCs were incubated with Env in the presence of mannan or incubated with Env-D368R to isolate degradation via CD4 and CLR-mediated uptake pathways respectively. Leupeptin was also included in these experiments as a control. Data here are the mean ± SD of 2 donors.

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

    Article Title: Human Plasmacytoid Dendritic Cells Efficiently Capture HIV-1 Envelope Glycoproteins via CD4 for Antigen Presentation

    doi: 10.4049/jimmunol.1202489

    Figure Lengend Snippet: Both CLR-mediated and CD4-mediated endocytosis of Env result in similar kinetics of antigen processing Cells were incubated with 10µg/mL FITC-labeled Env or HA for 90 min at 4°C or 37°C, then washed twice and cultured at 4°C or 37°C for up to 24h. In some cases, MDCs were pre-incubated and the experiment carried out in the presence of 1mM leupeptin (Leu) to inhibit proteolytic degradation. Samples were then stained with DC markers for analysis by flow cytometry and gated on HLA-DR + /CD11c + (MDCs), CD123 + (PDCs), CD1a + (LCs) or HLA-DR + (DDCs). A) Representative MDC donor showing the percentage of positive cells. B) The MFI for each timepoint was normalised to the 0 min 37°C control for each Ag and the data shown here are the mean ± SD for for 3–5 donors for Env and 1 donor for HA. C) Alternatively, MDCs were incubated with Env in the presence of mannan or incubated with Env-D368R to isolate degradation via CD4 and CLR-mediated uptake pathways respectively. Leupeptin was also included in these experiments as a control. Data here are the mean ± SD of 2 donors.

    Article Snippet: To retard degradation of antigens, cells were pre-incubated with, and the experiment and washes carried out in, the presence of a protease inhibitor 1mM Leupeptin (Roche).

    Techniques: Incubation, Labeling, Cell Culture, Staining, Flow Cytometry, Cytometry