protease inhibitor mix  (Thermo Fisher)

 
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    Name:
    E 64 Protease Inhibitor
    Description:
    Thermo Scientific E 64 Protease Inhibitor irreversably inhibits cysteine proteases Thermo Scientific Protease Inhibitors are small packages of individual protease inhibitor peptides and compounds for customized formulation or modification of protease inhibitor cocktails Features of Thermo Scientific E 64 Protease Inhibitor • High quality reagents in convenient and affordable package sizes • Purchase individually to prepare customized protease inhibitor cocktails • Supplement Halt Protease Inhibitor Cocktails whose formulations are fully disclosed to achieve desired concentrations of specific component reagents The individual reagents include AEBSF aprotinin bestatin E64 leupeptin pepstatin A and PMSF Although Thermo Scientific Halt Protease Inhibitor Cocktails provide convenient and optimized broad spectrum protease inhibition for routine cell lysis and assay needs certain applications require a more customized approach By offering the individual high quality reagents used to make our cocktail products we provide you with the tools needed to apply a protease inhibitor individually supplement a cocktail or combine several components to make customized protease inhibitor cocktails for specialized applications Related Products Pierce Protease and Phosphatase Inhibitor Mini Tablets EDTA Free Pierce Protease Inhibitor Mini Tablets EDTA Free
    Catalog Number:
    78434
    Price:
    None
    Applications:
    Cell Lysis & Fractionation|Protease and Phosphatase Inhibition|Protein Biology|Protein Purification & Isolation
    Category:
    Lab Reagents and Chemicals
    Buy from Supplier


    Structured Review

    Thermo Fisher protease inhibitor mix
    Thermo Scientific E 64 Protease Inhibitor irreversably inhibits cysteine proteases Thermo Scientific Protease Inhibitors are small packages of individual protease inhibitor peptides and compounds for customized formulation or modification of protease inhibitor cocktails Features of Thermo Scientific E 64 Protease Inhibitor • High quality reagents in convenient and affordable package sizes • Purchase individually to prepare customized protease inhibitor cocktails • Supplement Halt Protease Inhibitor Cocktails whose formulations are fully disclosed to achieve desired concentrations of specific component reagents The individual reagents include AEBSF aprotinin bestatin E64 leupeptin pepstatin A and PMSF Although Thermo Scientific Halt Protease Inhibitor Cocktails provide convenient and optimized broad spectrum protease inhibition for routine cell lysis and assay needs certain applications require a more customized approach By offering the individual high quality reagents used to make our cocktail products we provide you with the tools needed to apply a protease inhibitor individually supplement a cocktail or combine several components to make customized protease inhibitor cocktails for specialized applications Related Products Pierce Protease and Phosphatase Inhibitor Mini Tablets EDTA Free Pierce Protease Inhibitor Mini Tablets EDTA Free
    https://www.bioz.com/result/protease inhibitor mix/product/Thermo Fisher
    Average 97 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor mix - by Bioz Stars, 2021-02
    97/100 stars

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    Related Articles

    Protease Inhibitor:

    Article Title: eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy
    Article Snippet: .. PpIX degradation assay HepG2 cells were lysed by nondenaturing lysis buffer (Cell Signaling Technology Inc, Danvers, MA, USA) containing protease inhibitor PMSF (phenylmethylsulfonyl fluoride, 1 mM, Life Technologies) on ice for 10 min. .. The cell lysate was mixed with PpIX (1 μg/ml) and eEF1A1 (1.3 μg/ml, Abnova).

    Protein Extraction:

    Article Title: BH3 mimetic Obatoclax (GX15-070) mediates mitochondrial stress predominantly via MCL-1 inhibition and induces autophagy-dependent necroptosis in human oral cancer cells
    Article Snippet: .. Protein extraction and western blotting The cells were harvested and lysed in cell lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40) containing protease inhibitors (Fermentas, Canada). .. The cell lysates were incubated on ice for 30 minutes followed by centrifugation and the supernatants were subjected to protein estimation by Bradford assay (BioRad, USA).

    Cell Culture:

    Article Title: Upregulation of scavenger receptor B1 is required for steroidogenic and non-steroidogenic cholesterol metabolism in prostate cancer
    Article Snippet: .. BLT-1 (ChemBridge, San Diego, CA), a selective inhibitor of cholesteryl ester transfer through SR-B1 , or dimethyl sulfoxide (DMSO, vehicle), was added to cells cultured in phenol red-free media supplemented with 5% charcoal-dextran stripped FBS (CSS, Invitrogen) at the indicated final concentrations. ..

    Incubation:

    Article Title: Combination of cisplatin and bromelain exerts synergistic cytotoxic effects against breast cancer cell line MDA-MB-231 in vitro
    Article Snippet: .. Following an incubation period of 24 or 48 h, the cells were washed with PBS and lysed in RIPA lysis buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, 1% deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS)] containing protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). .. The extracted proteins (20–60 µg) were separated by electrophoresis on SDS–polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad) and probed with respective primary antibodies against Beta-actin, Bax and Bcl-2 (Abcam, Cambridge, Massachusetts, USA).

    Article Title: The Myopic-Ubpy-Hrs nexus enables endosomal recycling of Frizzled
    Article Snippet: .. E64 and MG132 treatment of wing imaginal disks In some experiments, third-instar wing disks were dissected and incubated at 25°C for 4 h in complete Drosophila insect cell medium supplemented with 2% heat-inactivated fetal bovine serum (FBS; Invitrogen), supplemented with either lysosomal (E64 [50 μM; Sigma-Aldrich]) or proteasomal (MG132 [50 μM; Sigma-Aldrich, St. Louis, MO]) inhibitors before fixation. .. Cell culture, transfection, transcription assays, and immunohistochemistry L Wnt-3a cells (Ramanuj Dasgupta) and L cells were cultured at 37°C in DMEM supplemented with 10% FBS (Invitrogen).

    other:

    Article Title: Increased Expression of Elastolytic Cysteine Proteases, Cathepsins S and K, in the Neointima of Balloon-Injured Rat Carotid Arteries
    Article Snippet: FITC-labeled type I collagen and E64 were from Molecular Probes (Eugene, Oregon).

    Degradation Assay:

    Article Title: eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy
    Article Snippet: .. PpIX degradation assay HepG2 cells were lysed by nondenaturing lysis buffer (Cell Signaling Technology Inc, Danvers, MA, USA) containing protease inhibitor PMSF (phenylmethylsulfonyl fluoride, 1 mM, Life Technologies) on ice for 10 min. .. The cell lysate was mixed with PpIX (1 μg/ml) and eEF1A1 (1.3 μg/ml, Abnova).

    Western Blot:

    Article Title: BH3 mimetic Obatoclax (GX15-070) mediates mitochondrial stress predominantly via MCL-1 inhibition and induces autophagy-dependent necroptosis in human oral cancer cells
    Article Snippet: .. Protein extraction and western blotting The cells were harvested and lysed in cell lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40) containing protease inhibitors (Fermentas, Canada). .. The cell lysates were incubated on ice for 30 minutes followed by centrifugation and the supernatants were subjected to protein estimation by Bradford assay (BioRad, USA).

    Article Title: Pharmacological targeting of apelin impairs glioblastoma growth
    Article Snippet: .. Western blots Following stimulation with the relevant treatment, cells were collected and washed in PBS before lysis at 4°C with TNT buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton™ X-100, 1% IGEPAL®) supplemented with protease inhibitors (ThermoFisher Scientific). .. Equal amounts of protein were loaded on tris-glycine gels and transferred onto nitrocellulose membranes (GE Healthcare).

    Article Title: Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair
    Article Snippet: .. Western blottingProtein was extracted for iPSMN cultures at day 30 using RIPA buffer with protease inhibitors and combined with Sample Reducing Agent and LDS sample buffer (Thermo Fisher Scientific). .. Samples were not heated and run in precast NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific) at 100V for 100 minutes.

    Lysis:

    Article Title: BH3 mimetic Obatoclax (GX15-070) mediates mitochondrial stress predominantly via MCL-1 inhibition and induces autophagy-dependent necroptosis in human oral cancer cells
    Article Snippet: .. Protein extraction and western blotting The cells were harvested and lysed in cell lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40) containing protease inhibitors (Fermentas, Canada). .. The cell lysates were incubated on ice for 30 minutes followed by centrifugation and the supernatants were subjected to protein estimation by Bradford assay (BioRad, USA).

    Article Title: eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy
    Article Snippet: .. PpIX degradation assay HepG2 cells were lysed by nondenaturing lysis buffer (Cell Signaling Technology Inc, Danvers, MA, USA) containing protease inhibitor PMSF (phenylmethylsulfonyl fluoride, 1 mM, Life Technologies) on ice for 10 min. .. The cell lysate was mixed with PpIX (1 μg/ml) and eEF1A1 (1.3 μg/ml, Abnova).

    Article Title: Pharmacological targeting of apelin impairs glioblastoma growth
    Article Snippet: .. Western blots Following stimulation with the relevant treatment, cells were collected and washed in PBS before lysis at 4°C with TNT buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton™ X-100, 1% IGEPAL®) supplemented with protease inhibitors (ThermoFisher Scientific). .. Equal amounts of protein were loaded on tris-glycine gels and transferred onto nitrocellulose membranes (GE Healthcare).

    Article Title: Combination of cisplatin and bromelain exerts synergistic cytotoxic effects against breast cancer cell line MDA-MB-231 in vitro
    Article Snippet: .. Following an incubation period of 24 or 48 h, the cells were washed with PBS and lysed in RIPA lysis buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, 1% deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS)] containing protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). .. The extracted proteins (20–60 µg) were separated by electrophoresis on SDS–polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad) and probed with respective primary antibodies against Beta-actin, Bax and Bcl-2 (Abcam, Cambridge, Massachusetts, USA).

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  • 90
    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: 90/100, 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 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    90
    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: 90/100, 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 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    erythrosin b - by Bioz Stars, 2021-02
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    93
    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: 93/100, 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 93 stars, based on 1 article reviews
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    97
    Thermo Fisher transfection mixture
    Protein expression and trypsin-mediated cleavage of MERS-CoV S protein WT and mutants. (A) Plasmid DNA encoding MERS-CoV S protein WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of <t>transfection,</t> as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B and C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection, and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full-length S proteins are visible at approximately 250 kDa. S1/S2-cleaved S proteins are visible at approximately 115 kDa.
    Transfection Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 346 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transfection mixture/product/Thermo Fisher
    Average 97 stars, based on 346 article reviews
    Price from $9.99 to $1999.99
    transfection mixture - by Bioz Stars, 2021-02
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    Image Search Results


    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

    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

    Protein expression and trypsin-mediated cleavage of MERS-CoV S protein WT and mutants. (A) Plasmid DNA encoding MERS-CoV S protein WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B and C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection, and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full-length S proteins are visible at approximately 250 kDa. S1/S2-cleaved S proteins are visible at approximately 115 kDa.

    Journal: Journal of Virology

    Article Title: Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

    doi: 10.1128/JVI.00426-20

    Figure Lengend Snippet: Protein expression and trypsin-mediated cleavage of MERS-CoV S protein WT and mutants. (A) Plasmid DNA encoding MERS-CoV S protein WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B and C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection, and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full-length S proteins are visible at approximately 250 kDa. S1/S2-cleaved S proteins are visible at approximately 115 kDa.

    Article Snippet: After 20 minutes, 2 ml cDMEM was added to the transfection mixture.

    Techniques: Expressing, Plasmid Preparation, Transfection, Protease Inhibitor, Concentration Assay, Isolation, SDS Page, Western Blot, Mutagenesis

    Immunofluorescence assay of MERS-CoV S protein WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding the respective MERS-CoV S protein variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleave MERS-CoV S proteins for fusion, no further protease treatment was needed to induce syncytium formation. WT + furin inhibitor (FI) indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection to block fusion. Syncytia were visualized using immunofluorescence microscopy by staining the MERS-CoV S protein with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI; in blue). Images were taken at a magnification of ×25. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted, and the average number of nuclei per syncytium was calculated. Error bars represent standard deviations ( n = 9). Statistical analysis was performed using an unpaired Student’s t test comparing the WT against each of the respective mutant *, P > 0.5; **, P > 0.05; ***, P > 0.005.

    Journal: Journal of Virology

    Article Title: Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

    doi: 10.1128/JVI.00426-20

    Figure Lengend Snippet: Immunofluorescence assay of MERS-CoV S protein WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding the respective MERS-CoV S protein variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleave MERS-CoV S proteins for fusion, no further protease treatment was needed to induce syncytium formation. WT + furin inhibitor (FI) indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection to block fusion. Syncytia were visualized using immunofluorescence microscopy by staining the MERS-CoV S protein with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI; in blue). Images were taken at a magnification of ×25. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted, and the average number of nuclei per syncytium was calculated. Error bars represent standard deviations ( n = 9). Statistical analysis was performed using an unpaired Student’s t test comparing the WT against each of the respective mutant *, P > 0.5; **, P > 0.05; ***, P > 0.005.

    Article Snippet: After 20 minutes, 2 ml cDMEM was added to the transfection mixture.

    Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Binding Assay, Protease Inhibitor, Concentration Assay, Blocking Assay, Microscopy, Staining, Mutagenesis