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Enzo Biochem filipin iii
Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), <t>filipin</t> <t>III</t> (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m
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1) Product Images from "Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity"

Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.459

Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), filipin III (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m
Figure Legend Snippet: Transduction of exogenously added CysC into N2a cells. ( a ) Transduction and localization of CysC to lysosomes in N2a cells. FITC-labeled CysC or bovine serum albumin (1 μ M) was added to N2a cells for 3 h, and the cells were then briefly treated by LysoTracker and analyzed by confocal microscopy. ( b ) Immunoblotting detection of transduced full-length CysC in purified lysosomal fractions. Biotin-conjugated transduced CysC was detected by horseradish peroxidase-streptavidin. Immunoblot using anti-LAMP-2 antibody indicates enrichment of lysosome. ( c ) Clathrin-dependent transduction of CysC. N2a cells were pre-treated with chlorpromazine (25 μ M), filipin III (5 mg/ml) or 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (25 μ M) for 1 h. Then, the cells were incubated with FITC-labeled CysC for 1 h, and analyzed by confocal microscopy. Scale bars: 25 μ m

Techniques Used: Transduction, Labeling, Confocal Microscopy, Purification, Incubation

Related Articles

Transduction:

Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity
Article Snippet: Paragraph title: Examination of CysC transduction into cells with fluorescence microscopy ... The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μ M chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μ M 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μ M FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components.

Transfection:

Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity
Article Snippet: The medium was replaced with the differentiation medium containing 1 μ M FITC-CysC or 300 nM rapamycin at 6 h after the transfection of pcDNA3.3-SOD1 or pAcGFP-N1-SOD1. .. The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μ M chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μ M 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μ M FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components.

Confocal Microscopy:

Article Title: Human SCARB2-Mediated Entry and Endocytosis of EV71
Article Snippet: The slides were then mounted with a coverslip and the cells observed in the same field of fluorescent and visible pictures was taken by the confocal microscopy (Leica TCS SP5 II). .. The following compounds used as inhibitors were purchased from Sigma-Aldrich, St Louis, MO, USA: genistein, filipin, methyl β-cyclodextrin (MβCD), chloroquine, and ammonium chloride (NH4 Cl). chlorpromazine (CPZ) was obtained from Alexis Biochemicals.

Fluorescence:

Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity
Article Snippet: Paragraph title: Examination of CysC transduction into cells with fluorescence microscopy ... The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μ M chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μ M 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μ M FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components.

Microscopy:

Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity
Article Snippet: Paragraph title: Examination of CysC transduction into cells with fluorescence microscopy ... The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μ M chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μ M 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μ M FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components.

Incubation:

Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity
Article Snippet: .. The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μ M chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μ M 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μ M FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components. .. The cells were washed with PBS twice and observed by confocal laser scanning microscopy.

Article Title: Human SCARB2-Mediated Entry and Endocytosis of EV71
Article Snippet: Cells were cooled to 15o C and then incubated with 5 µg/mL alexa fluor 594 CT-B conjugate (dissolved in 0.1% DMSO) (Invitrogen). .. The following compounds used as inhibitors were purchased from Sigma-Aldrich, St Louis, MO, USA: genistein, filipin, methyl β-cyclodextrin (MβCD), chloroquine, and ammonium chloride (NH4 Cl). chlorpromazine (CPZ) was obtained from Alexis Biochemicals.

Confocal Laser Scanning Microscopy:

Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity
Article Snippet: The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μ M chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μ M 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μ M FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components. .. The cells were washed with PBS twice and observed by confocal laser scanning microscopy.

Infection:

Article Title: Human SCARB2-Mediated Entry and Endocytosis of EV71
Article Snippet: Cells were treated with serial dilutions of MβCD for 30 minutes prior to the infection with EV71 as described above. .. The following compounds used as inhibitors were purchased from Sigma-Aldrich, St Louis, MO, USA: genistein, filipin, methyl β-cyclodextrin (MβCD), chloroquine, and ammonium chloride (NH4 Cl). chlorpromazine (CPZ) was obtained from Alexis Biochemicals.

Expressing:

Article Title: MCP-1/CCR2B-dependent loop upregulates MUC5AC and MUC5B in human airway epithelium
Article Snippet: In experiments designed to test the effects of MCP-1 on MUC5AC, MUC5B, and MCP-1 mRNA and protein expression, NHBE cells from at least three different lung donors and in duplicate wells for each experimental condition were apically exposed to PBS or human recombinant MCP-1 (50 ng/ml, R & D Systems, Minneapolis, MN). .. In experiments aimed at determining the signaling pathways activated by MCP-1, replicate cultures from n ≥ 3 different lung donors were pretreated with inhibitors for CCR2B [RS102895, 0.1 mM, 30 min ( )], 44/42 MAPK [U0126, 1 μM, 30 min ( )], caveolae integrity [methyl-β-cyclodextrin, 10 mM, 1 h ( ) or filipin, 5 μg/ml, 1 h ( )], Gq signaling [GP antagonist-2A, 10 μM, 1 h; Enzo, Plymouth Meeting, PA ( )], PLCβ [U-73122, 1 μM, 30 min ( )], PKC [Myr-RFARKGALRQKNV, 50 μM, 30 min ( )], or RhoA GTPase [cell-permeable C3 exoenzyme, 1 μg/ml, 30 min; Cytoskeleton, Denver, CO ( )] at 37°C.

Staining:

Article Title: Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity
Article Snippet: .. The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μ M chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μ M 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μ M FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components. .. The cells were washed with PBS twice and observed by confocal laser scanning microscopy.

Recombinant:

Article Title: MCP-1/CCR2B-dependent loop upregulates MUC5AC and MUC5B in human airway epithelium
Article Snippet: In experiments designed to test the effects of MCP-1 on MUC5AC, MUC5B, and MCP-1 mRNA and protein expression, NHBE cells from at least three different lung donors and in duplicate wells for each experimental condition were apically exposed to PBS or human recombinant MCP-1 (50 ng/ml, R & D Systems, Minneapolis, MN). .. In experiments aimed at determining the signaling pathways activated by MCP-1, replicate cultures from n ≥ 3 different lung donors were pretreated with inhibitors for CCR2B [RS102895, 0.1 mM, 30 min ( )], 44/42 MAPK [U0126, 1 μM, 30 min ( )], caveolae integrity [methyl-β-cyclodextrin, 10 mM, 1 h ( ) or filipin, 5 μg/ml, 1 h ( )], Gq signaling [GP antagonist-2A, 10 μM, 1 h; Enzo, Plymouth Meeting, PA ( )], PLCβ [U-73122, 1 μM, 30 min ( )], PKC [Myr-RFARKGALRQKNV, 50 μM, 30 min ( )], or RhoA GTPase [cell-permeable C3 exoenzyme, 1 μg/ml, 30 min; Cytoskeleton, Denver, CO ( )] at 37°C.

Inhibition:

Article Title: Human SCARB2-Mediated Entry and Endocytosis of EV71
Article Snippet: Paragraph title: Inhibition of endocytosis and endosomal acidification ... The following compounds used as inhibitors were purchased from Sigma-Aldrich, St Louis, MO, USA: genistein, filipin, methyl β-cyclodextrin (MβCD), chloroquine, and ammonium chloride (NH4 Cl). chlorpromazine (CPZ) was obtained from Alexis Biochemicals.

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    Enzo Biochem filipin
    MCP-1-induced mucin upregulation depends on 44/42 MAPK activation and requires caveolae integrity. Cells were exposed to apical PBS (control) or MCP-1 in the presence or the absence of the caveolae disruptors methy-β-cyclodextrin (MβCD) or <t>filipin.</t> A : samples ( n = 4 different lung donors) were analyzed for MUC5AC and MUC5B mRNA expression by qPCR. Results are expressed as fold change vs. controls. * P
    Filipin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MCP-1-induced mucin upregulation depends on 44/42 MAPK activation and requires caveolae integrity. Cells were exposed to apical PBS (control) or MCP-1 in the presence or the absence of the caveolae disruptors methy-β-cyclodextrin (MβCD) or filipin. A : samples ( n = 4 different lung donors) were analyzed for MUC5AC and MUC5B mRNA expression by qPCR. Results are expressed as fold change vs. controls. * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: MCP-1/CCR2B-dependent loop upregulates MUC5AC and MUC5B in human airway epithelium

    doi: 10.1152/ajplung.00292.2010

    Figure Lengend Snippet: MCP-1-induced mucin upregulation depends on 44/42 MAPK activation and requires caveolae integrity. Cells were exposed to apical PBS (control) or MCP-1 in the presence or the absence of the caveolae disruptors methy-β-cyclodextrin (MβCD) or filipin. A : samples ( n = 4 different lung donors) were analyzed for MUC5AC and MUC5B mRNA expression by qPCR. Results are expressed as fold change vs. controls. * P

    Article Snippet: In experiments aimed at determining the signaling pathways activated by MCP-1, replicate cultures from n ≥ 3 different lung donors were pretreated with inhibitors for CCR2B [RS102895, 0.1 mM, 30 min ( )], 44/42 MAPK [U0126, 1 μM, 30 min ( )], caveolae integrity [methyl-β-cyclodextrin, 10 mM, 1 h ( ) or filipin, 5 μg/ml, 1 h ( )], Gq signaling [GP antagonist-2A, 10 μM, 1 h; Enzo, Plymouth Meeting, PA ( )], PLCβ [U-73122, 1 μM, 30 min ( )], PKC [Myr-RFARKGALRQKNV, 50 μM, 30 min ( )], or RhoA GTPase [cell-permeable C3 exoenzyme, 1 μg/ml, 30 min; Cytoskeleton, Denver, CO ( )] at 37°C.

    Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction