protease inhibitor mix  (Thermo Fisher)


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    Structured Review

    Thermo Fisher protease inhibitor mix
    Protease Inhibitor Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protease inhibitor mix/product/Thermo Fisher
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor mix - by Bioz Stars, 2020-11
    99/100 stars

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    Related Articles

    Transfection:

    Article Title: Human wild-type superoxide dismutase 1 gene delivery to rat bone marrow stromal cells: its importance and potential future trends
    Article Snippet: .. Western blot The supernatant of the transfected cells was collected on ice, and protease inhibitor cocktail (Fermentas, Lithuania) was added immediately to the samples. ..

    Radio Immunoprecipitation:

    Article Title: ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation
    Article Snippet: .. Immunoblot analysis All samples were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM sodium orthovanadate, 1× sigma protease inhibitor cocktail) in ice for 30 min. Lysates were then quantified via Pierce® BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). .. In total, 10–50 μg of protein was separated by 10–15% SDS-PAGE and then transferred onto Immobilon® -P membrane (Millipore, Billerica, MA, USA).

    Homogenization:

    Article Title: P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer’s disease model
    Article Snippet: .. Protein extraction was performed by homogenization in PBS, pH 7.4, and 1% phosphatase inhibitor cocktail (Thermo Fisher) and 1% protease inhibitor cocktail (Thermo Fisher) using a ceramic bead homogenizer (Precellys; VWR). ..

    Protease Inhibitor:

    Article Title: HEXIM1-Tat chimera inhibits HIV-1 replication
    Article Snippet: .. After 48 hrs, the cells were washed using PBS, and lysed using RIPA buffer containing 150 mM KCl and a protease inhibitor mixture (Thermo Fisher Scientific). .. Lysates were precleared for 2 hrs at 4°C using protein G-sepharose beads (Life Technologyies, and the precleared lysates were incubated overnight at 4°C with 4 μg of the indicated primary Ab or control mouse IgG1 (MI10-102, Bethyl).

    Article Title: ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation
    Article Snippet: .. Immunoblot analysis All samples were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM sodium orthovanadate, 1× sigma protease inhibitor cocktail) in ice for 30 min. Lysates were then quantified via Pierce® BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). .. In total, 10–50 μg of protein was separated by 10–15% SDS-PAGE and then transferred onto Immobilon® -P membrane (Millipore, Billerica, MA, USA).

    Article Title: P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer’s disease model
    Article Snippet: .. Protein extraction was performed by homogenization in PBS, pH 7.4, and 1% phosphatase inhibitor cocktail (Thermo Fisher) and 1% protease inhibitor cocktail (Thermo Fisher) using a ceramic bead homogenizer (Precellys; VWR). ..

    Article Title:
    Article Snippet: .. Plates were placed on ice, PBS was aspirated, and cells were lysed by adding 50 μ l of lysis buffer (Pierce, Thermo Scientific Rockford, IL), supplemented with 1% phosphatase inhibitor cocktail 3, 100 nM okadaic acid, and 1% protease inhibitor (Pierce, Thermo Scientific) per well. .. Cells were scraped and centrifuged at 2000 rpm at 4°C for 3 minutes, and the supernatant was collected and prepared for SDS-PAGE following the NuPAGE protocol (Novex, Life Technologies Grand Island, NY).

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: .. LB medium, bovine serum albumin (BSA), NaCl, Triton X-100, glycerol, protease inhibitor cocktail, Tris-HCl (pH 7.5), and HisPur Ni-NTA agarose resin were purchased from Thermo Fisher Scientific (Waltham, MA). .. An In-Fusion HD cloning kit and Stellar competent cells were purchased from Clontech (Mountain View, CA).

    Article Title: Human wild-type superoxide dismutase 1 gene delivery to rat bone marrow stromal cells: its importance and potential future trends
    Article Snippet: .. Western blot The supernatant of the transfected cells was collected on ice, and protease inhibitor cocktail (Fermentas, Lithuania) was added immediately to the samples. ..

    Protein Extraction:

    Article Title: P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer’s disease model
    Article Snippet: .. Protein extraction was performed by homogenization in PBS, pH 7.4, and 1% phosphatase inhibitor cocktail (Thermo Fisher) and 1% protease inhibitor cocktail (Thermo Fisher) using a ceramic bead homogenizer (Precellys; VWR). ..

    BIA-KA:

    Article Title: ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation
    Article Snippet: .. Immunoblot analysis All samples were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM sodium orthovanadate, 1× sigma protease inhibitor cocktail) in ice for 30 min. Lysates were then quantified via Pierce® BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). .. In total, 10–50 μg of protein was separated by 10–15% SDS-PAGE and then transferred onto Immobilon® -P membrane (Millipore, Billerica, MA, USA).

    Western Blot:

    Article Title: Human wild-type superoxide dismutase 1 gene delivery to rat bone marrow stromal cells: its importance and potential future trends
    Article Snippet: .. Western blot The supernatant of the transfected cells was collected on ice, and protease inhibitor cocktail (Fermentas, Lithuania) was added immediately to the samples. ..

    Lysis:

    Article Title: ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation
    Article Snippet: .. Immunoblot analysis All samples were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM sodium orthovanadate, 1× sigma protease inhibitor cocktail) in ice for 30 min. Lysates were then quantified via Pierce® BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). .. In total, 10–50 μg of protein was separated by 10–15% SDS-PAGE and then transferred onto Immobilon® -P membrane (Millipore, Billerica, MA, USA).

    Article Title:
    Article Snippet: .. Plates were placed on ice, PBS was aspirated, and cells were lysed by adding 50 μ l of lysis buffer (Pierce, Thermo Scientific Rockford, IL), supplemented with 1% phosphatase inhibitor cocktail 3, 100 nM okadaic acid, and 1% protease inhibitor (Pierce, Thermo Scientific) per well. .. Cells were scraped and centrifuged at 2000 rpm at 4°C for 3 minutes, and the supernatant was collected and prepared for SDS-PAGE following the NuPAGE protocol (Novex, Life Technologies Grand Island, NY).

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    Thermo Fisher gene exp mlkl hs00930421 m1
    Study family pedigree and FA2H and <t>MLKL</t> rare variant genotype. a Pedigree of the study family showing the segregation of rare, small nucleotide deletions in FA2H and MLKL . Circles represent women, squares men, the solid symbol represent the clinically affected index patients (brother II-2 and brother II-3, who is the proband), open symbols represent unaffected family members, and dotted symbols indicate heterozygous individuals. A slash through a symbol represents a deceased person; in this family this is the father of the patients, I-1, who passed away in 2015 at the age of 95 years. b FA2H NM_024306.4:c.32_34del variant genotype of the study family members as determined by Sanger sequencing. Sequences in bold are the three nucleotides and corresponding amino acid that are deleted. c MLKL rs561839347 variant genotype of the study family members as determined by Sanger sequencing. Sequences in bold represent the four nucleotides that are deleted. Italicized amino acids are novel residues in the predicted protein due to the frameshift.
    Gene Exp Mlkl Hs00930421 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp mlkl hs00930421 m1/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    91
    Thermo Fisher 3xflag c fluc tagged ank1
    PF3D7_0402000 (D90c) interacts with <t>ANK1</t> and 4.1R in co-precipitation assays A) and C) Expression of proteins tagged with C-terminal fragment of firefly luciferase protein <t>(C-FLuc)</t> and 3XFLAG. ANK1 FG1 and 4.1R FG1 as fusions to C-FLuc-3XFLAG and C-FLuc-3XFLAG with no insert were in vitro translated in wheat germ extracts (WGE) and subjected to western blotting with anti-FLAG tag antibody. Molecular weight markers (sizes in kDa) are indicated at left. B) Co-affinity purification of erythrocyte ankyrin and 4.1R with MBP-His-tagged PF3D7_0402000 (MBP-D90c-His). C-FLuc-3XFLAG-tagged proteins from (A) were incubated with MBP-D90c-His and MBP-His and purified with amylose resin. The co-purifying proteins were subjected to SDS-PAGE analysis and western blotting using anti-FLAG antibody (top panel) and anti-MBP antibody (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left. D) Co-affinity purification of erythrocyte ankyrin and 4.1R with GST tagged-PF3D7_0402000 (GST-D90c). GST and GST-D90c were in vitro translated in WGE, incubated with C-FLuc-3XFLAG-tagged proteins from (C) and purified with GST beads. The co-purifying proteins were subjected to SDS-PAGE and western blotting using anti-FLAG (top panel) and anti-GST antibodies (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left.
    3xflag C Fluc Tagged Ank1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3xflag c fluc tagged ank1/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    91
    Thermo Fisher erythrosin b
    Inhibition of the NS2B-NS3 interactions and protease activity (A)  Dose-dependent inhibition of SLC upon binding of NLuc-NS2B 49-66  to GST-CLuc-NS3 by erythrosin B. N = 3. (B) Dose-response inhibitions of the DENV2 His-NS2B/His-MBP-NS3 protease activity by erythrosin B. N = 3. ( C ) Dose-response inhibitions of the ZIKV His-NS2B/GST-NS3 protease activity by erythrosin B. N = 3.
    Erythrosin B, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/erythrosin b/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
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    erythrosin b - by Bioz Stars, 2020-11
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    Image Search Results


    Study family pedigree and FA2H and MLKL rare variant genotype. a Pedigree of the study family showing the segregation of rare, small nucleotide deletions in FA2H and MLKL . Circles represent women, squares men, the solid symbol represent the clinically affected index patients (brother II-2 and brother II-3, who is the proband), open symbols represent unaffected family members, and dotted symbols indicate heterozygous individuals. A slash through a symbol represents a deceased person; in this family this is the father of the patients, I-1, who passed away in 2015 at the age of 95 years. b FA2H NM_024306.4:c.32_34del variant genotype of the study family members as determined by Sanger sequencing. Sequences in bold are the three nucleotides and corresponding amino acid that are deleted. c MLKL rs561839347 variant genotype of the study family members as determined by Sanger sequencing. Sequences in bold represent the four nucleotides that are deleted. Italicized amino acids are novel residues in the predicted protein due to the frameshift.

    Journal: Cell Death & Disease

    Article Title: A novel neurodegenerative spectrum disorder in patients with MLKL deficiency

    doi: 10.1038/s41419-020-2494-0

    Figure Lengend Snippet: Study family pedigree and FA2H and MLKL rare variant genotype. a Pedigree of the study family showing the segregation of rare, small nucleotide deletions in FA2H and MLKL . Circles represent women, squares men, the solid symbol represent the clinically affected index patients (brother II-2 and brother II-3, who is the proband), open symbols represent unaffected family members, and dotted symbols indicate heterozygous individuals. A slash through a symbol represents a deceased person; in this family this is the father of the patients, I-1, who passed away in 2015 at the age of 95 years. b FA2H NM_024306.4:c.32_34del variant genotype of the study family members as determined by Sanger sequencing. Sequences in bold are the three nucleotides and corresponding amino acid that are deleted. c MLKL rs561839347 variant genotype of the study family members as determined by Sanger sequencing. Sequences in bold represent the four nucleotides that are deleted. Italicized amino acids are novel residues in the predicted protein due to the frameshift.

    Article Snippet: Fibroblasts were stimulated with human interferon (IFN)-γ (PeproTech) for MLKL upregulation, and were also cultured with 25 μM of the protease inhibitor MG-132 (Sigma-Aldrich) or 2 μM of the MLKL inhibitor necrosulfonamide (NSA) (Millipore) for 24 h. Real-time quantitative PCR was performed using TaqMan® Gene Expression Assays MLKL_Hs00930421_m1 and GAPDH_Hs02758991_g1.

    Techniques: Variant Assay, Sequencing

    MLKL variant expression and effect on necroptosis. a MLKL protein expression in PBMCs of the sister II-1, unrelated controls C1–C5 and the brothers, II-3 and II-2. The molecular weight, in kilodaltons (kDa), of full-length MLKL and of the predicted, truncated MLKL variant (which was not detectable), relative to the marker, are denoted by the arrows. GAPDH was used as the loading control. b Relative expression of MLKL messenger RNA (mRNA) in PBMCs from controls (sister II-1, and unrelated controls C1–C5; shown in blue) and the patients (brothers II-3 and II-2; shown in red), assessed in triplicate. c MLKL protein expression in unstimulated fibroblasts (−) and fibroblasts stimulated with 1000 ng/ml of IFNγ for 24 h (+). Lysate of HEK-293 cells transiently transfected with the truncated MLKL variant protein was used as a control. d Relative expression and upregulation of MLKL mRNA upon stimulation of fibroblasts from controls (sister II-1 and unrelated control F1; depicted as blue triangles and diamonds, respectively), and the patients (brothers II-3 and II-2; depicted as red squares and circles, respectively) with varying concentrations of IFNγ for 24 h. Three independent experiments were performed. e Proportion of dying fibroblasts from the controls (sister II-1 and unrelated control F1; depicted as blue triangles and diamonds, respectively), and the patients (brothers II-3 and II-2; depicted as red squares and circles, respectively) over an 87-h period. Necroptosis sensitization was achieved by RIPK3 transduction in the presence of 5 ng/ml IFNγ and 50 μM zVAD, and cell death was measured based on the uptake of IncuCyte® Cytotox Red Reagent. Four independent experiments were performed. Blue and dark blue asterisks (*) denote a statistically significant difference ( P

    Journal: Cell Death & Disease

    Article Title: A novel neurodegenerative spectrum disorder in patients with MLKL deficiency

    doi: 10.1038/s41419-020-2494-0

    Figure Lengend Snippet: MLKL variant expression and effect on necroptosis. a MLKL protein expression in PBMCs of the sister II-1, unrelated controls C1–C5 and the brothers, II-3 and II-2. The molecular weight, in kilodaltons (kDa), of full-length MLKL and of the predicted, truncated MLKL variant (which was not detectable), relative to the marker, are denoted by the arrows. GAPDH was used as the loading control. b Relative expression of MLKL messenger RNA (mRNA) in PBMCs from controls (sister II-1, and unrelated controls C1–C5; shown in blue) and the patients (brothers II-3 and II-2; shown in red), assessed in triplicate. c MLKL protein expression in unstimulated fibroblasts (−) and fibroblasts stimulated with 1000 ng/ml of IFNγ for 24 h (+). Lysate of HEK-293 cells transiently transfected with the truncated MLKL variant protein was used as a control. d Relative expression and upregulation of MLKL mRNA upon stimulation of fibroblasts from controls (sister II-1 and unrelated control F1; depicted as blue triangles and diamonds, respectively), and the patients (brothers II-3 and II-2; depicted as red squares and circles, respectively) with varying concentrations of IFNγ for 24 h. Three independent experiments were performed. e Proportion of dying fibroblasts from the controls (sister II-1 and unrelated control F1; depicted as blue triangles and diamonds, respectively), and the patients (brothers II-3 and II-2; depicted as red squares and circles, respectively) over an 87-h period. Necroptosis sensitization was achieved by RIPK3 transduction in the presence of 5 ng/ml IFNγ and 50 μM zVAD, and cell death was measured based on the uptake of IncuCyte® Cytotox Red Reagent. Four independent experiments were performed. Blue and dark blue asterisks (*) denote a statistically significant difference ( P

    Article Snippet: Fibroblasts were stimulated with human interferon (IFN)-γ (PeproTech) for MLKL upregulation, and were also cultured with 25 μM of the protease inhibitor MG-132 (Sigma-Aldrich) or 2 μM of the MLKL inhibitor necrosulfonamide (NSA) (Millipore) for 24 h. Real-time quantitative PCR was performed using TaqMan® Gene Expression Assays MLKL_Hs00930421_m1 and GAPDH_Hs02758991_g1.

    Techniques: Variant Assay, Expressing, Molecular Weight, Marker, Transfection, Transduction

    PF3D7_0402000 (D90c) interacts with ANK1 and 4.1R in co-precipitation assays A) and C) Expression of proteins tagged with C-terminal fragment of firefly luciferase protein (C-FLuc) and 3XFLAG. ANK1 FG1 and 4.1R FG1 as fusions to C-FLuc-3XFLAG and C-FLuc-3XFLAG with no insert were in vitro translated in wheat germ extracts (WGE) and subjected to western blotting with anti-FLAG tag antibody. Molecular weight markers (sizes in kDa) are indicated at left. B) Co-affinity purification of erythrocyte ankyrin and 4.1R with MBP-His-tagged PF3D7_0402000 (MBP-D90c-His). C-FLuc-3XFLAG-tagged proteins from (A) were incubated with MBP-D90c-His and MBP-His and purified with amylose resin. The co-purifying proteins were subjected to SDS-PAGE analysis and western blotting using anti-FLAG antibody (top panel) and anti-MBP antibody (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left. D) Co-affinity purification of erythrocyte ankyrin and 4.1R with GST tagged-PF3D7_0402000 (GST-D90c). GST and GST-D90c were in vitro translated in WGE, incubated with C-FLuc-3XFLAG-tagged proteins from (C) and purified with GST beads. The co-purifying proteins were subjected to SDS-PAGE and western blotting using anti-FLAG (top panel) and anti-GST antibodies (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left.

    Journal: Molecular and biochemical parasitology

    Article Title: The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

    doi: 10.1016/j.molbiopara.2017.06.002

    Figure Lengend Snippet: PF3D7_0402000 (D90c) interacts with ANK1 and 4.1R in co-precipitation assays A) and C) Expression of proteins tagged with C-terminal fragment of firefly luciferase protein (C-FLuc) and 3XFLAG. ANK1 FG1 and 4.1R FG1 as fusions to C-FLuc-3XFLAG and C-FLuc-3XFLAG with no insert were in vitro translated in wheat germ extracts (WGE) and subjected to western blotting with anti-FLAG tag antibody. Molecular weight markers (sizes in kDa) are indicated at left. B) Co-affinity purification of erythrocyte ankyrin and 4.1R with MBP-His-tagged PF3D7_0402000 (MBP-D90c-His). C-FLuc-3XFLAG-tagged proteins from (A) were incubated with MBP-D90c-His and MBP-His and purified with amylose resin. The co-purifying proteins were subjected to SDS-PAGE analysis and western blotting using anti-FLAG antibody (top panel) and anti-MBP antibody (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left. D) Co-affinity purification of erythrocyte ankyrin and 4.1R with GST tagged-PF3D7_0402000 (GST-D90c). GST and GST-D90c were in vitro translated in WGE, incubated with C-FLuc-3XFLAG-tagged proteins from (C) and purified with GST beads. The co-purifying proteins were subjected to SDS-PAGE and western blotting using anti-FLAG (top panel) and anti-GST antibodies (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left.

    Article Snippet: GST or GST-D90c were mixed with 3XFLAG-C-FLuc tagged ANK1 and 4.1R, incubated with GST beads (Thermo Scientific) overnight at 4° C in presence of protease inhibitor (PI) cocktail (Roche), washed three times with PBS+0.1% TritonX-100, eluted by boiling in Laemmli SDS-PAGE sample loading buffer, and subjected to western blot analysis.

    Techniques: Expressing, Luciferase, In Vitro, Western Blot, FLAG-tag, Molecular Weight, Affinity Purification, Incubation, Purification, SDS Page

    PF3D7_0402000 (D90c) targets the 2nd folding domain of ANK1 A) Expression of proteins tagged with the C-terminal fragment of firefly luciferase protein (C-FLuc) and 3XFLAG. D1, D2 and D1+D2 subdomains of ANK1 as fusions to C-FLuc-3XFLAG and C-FLuc-3XFLAG with no insert were in vitro translated in wheat germ extracts (WGE) and subjected to SDS-PAGE and western blotting with anti-FLAG tag antibody. Molecular weight markers (sizes in kDa) are indicated at left. B) Co-affinity purification of ANK1 subdomains with MBP-D90c-His. C-FLuc-3XFLAG-tagged proteins from (A) were incubated with MBP-D90c-His and MBP-His and purified with amylose resin. The co-purifying proteins were subjected to SDS-PAGE and western blotting using anti- FLAG (top panel) and anti-MBP antibodies (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left.

    Journal: Molecular and biochemical parasitology

    Article Title: The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

    doi: 10.1016/j.molbiopara.2017.06.002

    Figure Lengend Snippet: PF3D7_0402000 (D90c) targets the 2nd folding domain of ANK1 A) Expression of proteins tagged with the C-terminal fragment of firefly luciferase protein (C-FLuc) and 3XFLAG. D1, D2 and D1+D2 subdomains of ANK1 as fusions to C-FLuc-3XFLAG and C-FLuc-3XFLAG with no insert were in vitro translated in wheat germ extracts (WGE) and subjected to SDS-PAGE and western blotting with anti-FLAG tag antibody. Molecular weight markers (sizes in kDa) are indicated at left. B) Co-affinity purification of ANK1 subdomains with MBP-D90c-His. C-FLuc-3XFLAG-tagged proteins from (A) were incubated with MBP-D90c-His and MBP-His and purified with amylose resin. The co-purifying proteins were subjected to SDS-PAGE and western blotting using anti- FLAG (top panel) and anti-MBP antibodies (bottom panel). Molecular weight markers (sizes in kDa) are indicated at left.

    Article Snippet: GST or GST-D90c were mixed with 3XFLAG-C-FLuc tagged ANK1 and 4.1R, incubated with GST beads (Thermo Scientific) overnight at 4° C in presence of protease inhibitor (PI) cocktail (Roche), washed three times with PBS+0.1% TritonX-100, eluted by boiling in Laemmli SDS-PAGE sample loading buffer, and subjected to western blot analysis.

    Techniques: Expressing, Luciferase, In Vitro, SDS Page, Western Blot, FLAG-tag, Molecular Weight, Affinity Purification, Incubation, Purification

    Inhibition of the NS2B-NS3 interactions and protease activity (A)  Dose-dependent inhibition of SLC upon binding of NLuc-NS2B 49-66  to GST-CLuc-NS3 by erythrosin B. N = 3. (B) Dose-response inhibitions of the DENV2 His-NS2B/His-MBP-NS3 protease activity by erythrosin B. N = 3. ( C ) Dose-response inhibitions of the ZIKV His-NS2B/GST-NS3 protease activity by erythrosin B. N = 3.

    Journal: Antiviral research

    Article Title: Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease

    doi: 10.1016/j.antiviral.2017.12.018

    Figure Lengend Snippet: Inhibition of the NS2B-NS3 interactions and protease activity (A) Dose-dependent inhibition of SLC upon binding of NLuc-NS2B 49-66 to GST-CLuc-NS3 by erythrosin B. N = 3. (B) Dose-response inhibitions of the DENV2 His-NS2B/His-MBP-NS3 protease activity by erythrosin B. N = 3. ( C ) Dose-response inhibitions of the ZIKV His-NS2B/GST-NS3 protease activity by erythrosin B. N = 3.

    Article Snippet: The DENV2 His-MBP-NS3 or each mutant (final concentration of 2.5 μM in 1x PBS) was mixed with erythrosin B to attain a 4.8 μM final concentration in 1.6% DMSO in the MicroAmp® Fast Optical 96-Well Reaction Plate (ThermoFisher Scientific).

    Techniques: Inhibition, Activity Assay, Binding Assay

    Inhibition of ZIKV in cells relevant to ZIKV ( A ) qRT-PCR analysis of inhibition of viral RNA from ZIKV infected A549 cells by erythrosin B.  (B)  Immunofluorescence assay (IFA) of inhibition of viral protein expression by erythrosin B, using pan-flavivirus anti-E 4G2 antibody (green) (ATCC). Nuclei (blue) was stained in all IFA assays by the Hoechst stain solution.  (C) ). At 48 h post infection, cells were washed, harvested, and protected by protease inhibitor cocktail prior to lysis by SDS-PAGE loading buffer. Upon incubation at 95 °C for 10 min, sample was subjected to Western blot analysis with anti-ZIKV NS3 (GTX133309, GeneTex, Inc.) and anti-GAPDH (CB1001, EMD Millipore) as primary antibodies. ( D,E ) Viral plaque reduction assay for ZIKV-infected HPECs ( D ) and iPSC-derived hNPCs ( E ) by erythrosin B. ***, p

    Journal: Antiviral research

    Article Title: Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease

    doi: 10.1016/j.antiviral.2017.12.018

    Figure Lengend Snippet: Inhibition of ZIKV in cells relevant to ZIKV ( A ) qRT-PCR analysis of inhibition of viral RNA from ZIKV infected A549 cells by erythrosin B. (B) Immunofluorescence assay (IFA) of inhibition of viral protein expression by erythrosin B, using pan-flavivirus anti-E 4G2 antibody (green) (ATCC). Nuclei (blue) was stained in all IFA assays by the Hoechst stain solution. (C) ). At 48 h post infection, cells were washed, harvested, and protected by protease inhibitor cocktail prior to lysis by SDS-PAGE loading buffer. Upon incubation at 95 °C for 10 min, sample was subjected to Western blot analysis with anti-ZIKV NS3 (GTX133309, GeneTex, Inc.) and anti-GAPDH (CB1001, EMD Millipore) as primary antibodies. ( D,E ) Viral plaque reduction assay for ZIKV-infected HPECs ( D ) and iPSC-derived hNPCs ( E ) by erythrosin B. ***, p

    Article Snippet: The DENV2 His-MBP-NS3 or each mutant (final concentration of 2.5 μM in 1x PBS) was mixed with erythrosin B to attain a 4.8 μM final concentration in 1.6% DMSO in the MicroAmp® Fast Optical 96-Well Reaction Plate (ThermoFisher Scientific).

    Techniques: Inhibition, Quantitative RT-PCR, Infection, Immunofluorescence, Expressing, Staining, Protease Inhibitor, Lysis, SDS Page, Incubation, Western Blot, Derivative Assay

    Inhibition of NS2B-NS3 protease complex via a non-competitive mechanism ( A,B ) Inhibition kinetic experiment of the DENV2 His-NS2B/His-MBP-NS3 ( A ) and the ZIKV His-NS2B/GST-NS3 ( B ) heterocomplexes by erythrosin B. Lineweaver–Burk plots of kinetics experimental data for inhibition of the His-NS2B/His-MBP-NS3 hetero protease complexes by erythrosin B. The DENV2 MBP-NS3 or ZIKV GST-NS3 (100 nM) was mixed with erythrosin B (3 μM or various concentrations) for 30 min. The DENV2 His-NS2B or ZIKV His-NS2B (1 μM) were added together with the Abz substrate at various concentrations (800 μM–25 μM in 2-fold dilutions). ( C ) Superimposition of bound EB (yellow) with the NS2B co-factor (cyan) of DENV3 (PDB: 3U1I). NS3 residues in contact with EB were in stick representation (grey). ( D ) Ribbon presentation of erythrosin B (green) docked into NS3pro of DENV3 (PDB: 3U1I). Key interaction residues are highlighted in stick presentation. Hydrogen bonds are shown in yellow dotted lines and π - π stacking is shown in blue line. ( E ) Ribbon presentation of erythrosin B (green) docked into NS3pro of ZIKV (PDB: 5LC0). The corresponding NS3pro β -strand hairpin loops on ZIKV are shown in green and grey. Key interaction residues are highlighted in stick presentation. Hydrogen bonds are also shown in yellow dotted lines and π - π stacking is shown in blue lines.

    Journal: Antiviral research

    Article Title: Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease

    doi: 10.1016/j.antiviral.2017.12.018

    Figure Lengend Snippet: Inhibition of NS2B-NS3 protease complex via a non-competitive mechanism ( A,B ) Inhibition kinetic experiment of the DENV2 His-NS2B/His-MBP-NS3 ( A ) and the ZIKV His-NS2B/GST-NS3 ( B ) heterocomplexes by erythrosin B. Lineweaver–Burk plots of kinetics experimental data for inhibition of the His-NS2B/His-MBP-NS3 hetero protease complexes by erythrosin B. The DENV2 MBP-NS3 or ZIKV GST-NS3 (100 nM) was mixed with erythrosin B (3 μM or various concentrations) for 30 min. The DENV2 His-NS2B or ZIKV His-NS2B (1 μM) were added together with the Abz substrate at various concentrations (800 μM–25 μM in 2-fold dilutions). ( C ) Superimposition of bound EB (yellow) with the NS2B co-factor (cyan) of DENV3 (PDB: 3U1I). NS3 residues in contact with EB were in stick representation (grey). ( D ) Ribbon presentation of erythrosin B (green) docked into NS3pro of DENV3 (PDB: 3U1I). Key interaction residues are highlighted in stick presentation. Hydrogen bonds are shown in yellow dotted lines and π - π stacking is shown in blue line. ( E ) Ribbon presentation of erythrosin B (green) docked into NS3pro of ZIKV (PDB: 5LC0). The corresponding NS3pro β -strand hairpin loops on ZIKV are shown in green and grey. Key interaction residues are highlighted in stick presentation. Hydrogen bonds are also shown in yellow dotted lines and π - π stacking is shown in blue lines.

    Article Snippet: The DENV2 His-MBP-NS3 or each mutant (final concentration of 2.5 μM in 1x PBS) was mixed with erythrosin B to attain a 4.8 μM final concentration in 1.6% DMSO in the MicroAmp® Fast Optical 96-Well Reaction Plate (ThermoFisher Scientific).

    Techniques: Inhibition