protease inhibitors  (Millipore)


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    Structured Review

    Millipore protease inhibitors
    Protease Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 546 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protease inhibitors/product/Millipore
    Average 99 stars, based on 546 article reviews
    Price from $9.99 to $1999.99
    protease inhibitors - by Bioz Stars, 2020-04
    99/100 stars

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    Centrifugation:

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: After an incubation period of 72 h, cells were collected by centrifugation at 2500 rpm for 7 min, washed twice with PBS and finally resuspended in 300 μl of PBS containing 2.5 μM of annexin V-FITC (Thermo Fisher, MA, USA) and 1 μg ml-1 propidium iodide (Sigma-Aldrich) for 10 min at 37°C in the dark. .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    Synthesized:

    Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy
    Article Snippet: The cells were starved in starvation medium (culture medium containing 0.5% fetal bovine serum) for 24 hours at 37°C and then treated under serum starvation conditions for 2 hours with cetuximab and the synthesized SPIONs prior to stimulation with 30 ng/mL epidermal growth factor for 30 minutes at 37°C. .. After washing once with cold 1× PBS, the cell pellet was lysed with RIPA lysis buffer (1× PBS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride, 0.25% sodium deoxycholate, 1% NP-40, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride) with 1% freshly added SigmaFAST™ protease inhibitor cocktail (s8830, Sigma-Aldrich, St Louis, MO, USA).

    Cell Fractionation:

    Article Title: Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane
    Article Snippet: Paragraph title: Cell fractionation and immunoblotting. ... To assay for an association with detergent-resistant membranes (see ), cells were washed once in PBS and lysed at either 4°C or 37°C in PBS with 1% Triton X-100 and protease inhibitors (SigmaFAST cocktail; Sigma-Aldrich) as described previously ( ).

    Cytometry:

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: Apoptotic cells were determined by analyzing 60,000 ungated cells by using a flow cytometer (Coulter Cytoflex, Beckman, CA, USA) and the FlowJo software. .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    Blocking Assay:

    Article Title: An essential contractile ring protein controls cell division in Plasmodium falciparum
    Article Snippet: Quantitative western blotting Parasite pellets were purified by lysing RBCs in 0.05% saponin in PBS with protease inhibitors (SigmaFast Protease Inhibitor Cocktail, Sigma) and boiled in Laemmli buffer. .. Membranes were blocked in Licor Odyssey blocking buffer, incubated with primary antibody diluted (1:1000 for anti-V5 or 1:2000 for anti-PfLDH) in blocking buffer, and then incubated with secondary antibodies diluted in blocking buffer.

    Incubation:

    Article Title: MBNL splicing activity depends on RNA binding site structural context
    Article Snippet: Immunoblotting HeLa cells were lysed with RIPA buffer supplemented with SIGMAFAST Protease Inhibitor Cocktail (Sigma). .. Membranes were blocked for 1 h with 5% skim milk in PBST buffer (phosphate-buffered saline (PBS), 0.1% Tween-20) and incubated with a primary antibody against eGFP (1:1000, Santa Cruz cat. no. sc-8334) or human GAPDH (1:1000, Santa Cruz cat. no. sc-47724).

    Article Title: An essential contractile ring protein controls cell division in Plasmodium falciparum
    Article Snippet: Quantitative western blotting Parasite pellets were purified by lysing RBCs in 0.05% saponin in PBS with protease inhibitors (SigmaFast Protease Inhibitor Cocktail, Sigma) and boiled in Laemmli buffer. .. Membranes were blocked in Licor Odyssey blocking buffer, incubated with primary antibody diluted (1:1000 for anti-V5 or 1:2000 for anti-PfLDH) in blocking buffer, and then incubated with secondary antibodies diluted in blocking buffer.

    Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy
    Article Snippet: After washing once with cold 1× PBS, the cell pellet was lysed with RIPA lysis buffer (1× PBS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride, 0.25% sodium deoxycholate, 1% NP-40, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride) with 1% freshly added SigmaFAST™ protease inhibitor cocktail (s8830, Sigma-Aldrich, St Louis, MO, USA). .. The membranes were then incubated with primary antibodies against phospho-EGFR (Tyr1173; 1:2,000; Novus Biologi-cals Inc, Littleton, CO, USA) and β-actin (1:10,000; GeneTex Inc, Irvine, CA, USA), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (1:10,000; Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

    Article Title: Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa
    Article Snippet: For collection of intestinal lavage (IL) samples, small intestine was removed and carefully filled with 3 mL of PBS containing Sigmafast Protease Inhibitor Cocktail (Sigma, USA) using a syringe. .. After 10 min incubation, solution was transferred into the 1.5 mL conical tubes (Eppendorf, Germany).

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich). ..

    Expressing:

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: To investigate the effect of nutlin-3a and of Nut-Mag-SLNs on the expression of p53 and its downstream proteins (e.g., MDM2 and p21), western blotting analysis was performed. .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    BIA-KA:

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich). .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    Modification:

    Article Title: Transcriptomic profiles of aging in purified human immune cells
    Article Snippet: .. Pellet was resuspended in 100 μl modified 4× Laemmli buffer [ ] (4% SDS, 250 mM Tris HCl, no glycerol, no bromophenol blue, no β-mercaptoethanol) mixed 1:1 with 8 M Urea [ ], with SigmaFAST Protease Inhibitor Cocktail Tablet (Sigma-Aldrich, St. Louis, MO). .. Samples were warmed to 55°C, and sonicated 4 × 30 seconds in a Bioruptor (Diagenode, Denville, NJ).

    Article Title: Surfen, a proteoglycan binding agent, reduces inflammation but inhibits remyelination in murine models of Multiple Sclerosis
    Article Snippet: .. Acetic acid, ammonium sulphamate (H6 N2 O3 S), bovine serum albumin (BSA), brefeldin A from penicillium , complete Freund’s adjuvant (CFA) containing mycobacterium tuberculosis H37RA (10 mg/ml), concanavalin A, dimethyl sulfoxide (DMSO), chondroitinase ABC, cysteine hydrochloric acid (HCL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), eriochrome cyanine, Dulbecco’s Modified Eagle’s Medium (DMEM), 10% buffered formalin, Griess reagent for nitrite, ketoprofen, L-α-Lysophosphatidylcholine (lysolecithin) from egg yolk, phosphate buffered saline (PBS; pH 7.4), pertussis toxin from Bordetella pertussis, Roswell Park Memorial Institute 1640 medium (RPMI), heparinase-III, ionomycin, bacterial lipopolysaccharide (LPS) from E. coli 0111:B4, papain from papaya latex, phorbol 12-myristate 13-acetate (PMA), sodium azide (NaN3 ), sodium citrate, sodium nitrite (NaNO2 ), sodium phosphate (NaH2 PO4 ), SIGMA FAST ® protease inhibitor cocktail tablets, surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide hydrate), and Triton-X-100 were obtained from Sigma-Aldrich Canada (Oakville, ON). .. Fetal bovine serum (FBS), 10,000 U/ml penicillin/10,000 μg/mL streptomycin solution, 200 mM L-glutamine, 1 M 4-(2-hydroyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, and 0.4% trypan blue dye solution were obtained from Invitrogen Canada (Oakville, ON).

    Western Blot:

    Article Title: MBNL splicing activity depends on RNA binding site structural context
    Article Snippet: Immunoblotting HeLa cells were lysed with RIPA buffer supplemented with SIGMAFAST Protease Inhibitor Cocktail (Sigma). .. Anti-rabbit (1:20 000, Sigma cat. no. A9169) and anti-mouse (1:2000, Millipore cat. no. 12-349) secondary antibodies were conjugated with horseradish peroxidase and detected using the Pierce ECL Plus Western Blotting Substrate detection kit (Thermo Scientific).

    Article Title: An essential contractile ring protein controls cell division in Plasmodium falciparum
    Article Snippet: .. Quantitative western blotting Parasite pellets were purified by lysing RBCs in 0.05% saponin in PBS with protease inhibitors (SigmaFast Protease Inhibitor Cocktail, Sigma) and boiled in Laemmli buffer. ..

    Article Title: Transcriptomic profiles of aging in purified human immune cells
    Article Snippet: Paragraph title: MCL1 and MRPS12 protein extraction and western blotting ... Pellet was resuspended in 100 μl modified 4× Laemmli buffer [ ] (4% SDS, 250 mM Tris HCl, no glycerol, no bromophenol blue, no β-mercaptoethanol) mixed 1:1 with 8 M Urea [ ], with SigmaFAST Protease Inhibitor Cocktail Tablet (Sigma-Aldrich, St. Louis, MO).

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: To investigate the effect of nutlin-3a and of Nut-Mag-SLNs on the expression of p53 and its downstream proteins (e.g., MDM2 and p21), western blotting analysis was performed. .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    Article Title: The Distribution of Cholinergic Neurons, and their Co-localization with FMRFamide in Central and Peripheral Neurons of the Spider Cupiennius salei
    Article Snippet: Paragraph title: Western blot analysis ... Spider brain tissue was placed in a 1 ml glass tissue grinder containing ice-cold spider saline with protease inhibitor (Sigma S-8830) at a dilution of 1 part protease inhibitor to 10 parts spider saline.

    Article Title: Extracellular Vesicles Released by Oxidatively Injured or Intact C2C12 Myotubes Promote Distinct Responses Converging toward Myogenesis
    Article Snippet: Paragraph title: 4.6. Western Blotting Analysis ... The protein pellet was dissolved in ISOT buffer (8 M urea, 4% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate), 65 mM DTE (1,4-Dithioerythritol), 40 mM Tris base and added with SIGMAFAST™ Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy)) and sonicated for 5 s on ice.

    Flow Cytometry:

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: Apoptotic cells were determined by analyzing 60,000 ungated cells by using a flow cytometer (Coulter Cytoflex, Beckman, CA, USA) and the FlowJo software. .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    Protease Inhibitor:

    Article Title: MBNL splicing activity depends on RNA binding site structural context
    Article Snippet: .. Immunoblotting HeLa cells were lysed with RIPA buffer supplemented with SIGMAFAST Protease Inhibitor Cocktail (Sigma). .. Samples were heated to 95°C for 5 min, separated on 10% SDS polyacrylamide gels and transferred to nitrocellulose (Protran BA 85, Whatman) using a wet transfer apparatus (1 h, 100 V, 4°C).

    Article Title: Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor
    Article Snippet: .. Aliquots of homogenized membrane preparations, corresponding to 100 μg of protein, were centrifuged at 50,000 g at 4 °C for 15 min, and the pellets were re-suspended in 50 mM Tris, pH 7.4, supplemented with 1 mM EDTA and protease inhibitors (SIGMAFAST Protease Inhibitor cocktail tablets, Sigma) at a protein concentration of 1,600 μg ml−1 . .. Membrane homogenates (15 μl) were processed and subjected to immunoblotting as described previously with the following modifications: blotting onto the nitrocellulose membrane was performed at 60 mA for 60 min. Primary antibody ANTI-FLAG M1 from mouse (Sigma, order no. F3040, lot SLBK1592V) was diluted 1:500.

    Article Title: An essential contractile ring protein controls cell division in Plasmodium falciparum
    Article Snippet: .. Quantitative western blotting Parasite pellets were purified by lysing RBCs in 0.05% saponin in PBS with protease inhibitors (SigmaFast Protease Inhibitor Cocktail, Sigma) and boiled in Laemmli buffer. ..

    Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy
    Article Snippet: .. After washing once with cold 1× PBS, the cell pellet was lysed with RIPA lysis buffer (1× PBS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride, 0.25% sodium deoxycholate, 1% NP-40, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride) with 1% freshly added SigmaFAST™ protease inhibitor cocktail (s8830, Sigma-Aldrich, St Louis, MO, USA). .. The resulting mixture was centrifuged at 14,000× g for 10 minutes at 4°C, and the protein concentration in the supernatant was measured using Bradford reagents (Sigma-Aldrich).

    Article Title: Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa
    Article Snippet: .. For collection of intestinal lavage (IL) samples, small intestine was removed and carefully filled with 3 mL of PBS containing Sigmafast Protease Inhibitor Cocktail (Sigma, USA) using a syringe. .. After 10 min incubation, solution was transferred into the 1.5 mL conical tubes (Eppendorf, Germany).

    Article Title: Transcriptomic profiles of aging in purified human immune cells
    Article Snippet: .. Pellet was resuspended in 100 μl modified 4× Laemmli buffer [ ] (4% SDS, 250 mM Tris HCl, no glycerol, no bromophenol blue, no β-mercaptoethanol) mixed 1:1 with 8 M Urea [ ], with SigmaFAST Protease Inhibitor Cocktail Tablet (Sigma-Aldrich, St. Louis, MO). .. Samples were warmed to 55°C, and sonicated 4 × 30 seconds in a Bioruptor (Diagenode, Denville, NJ).

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich). ..

    Article Title: Surfen, a proteoglycan binding agent, reduces inflammation but inhibits remyelination in murine models of Multiple Sclerosis
    Article Snippet: .. Acetic acid, ammonium sulphamate (H6 N2 O3 S), bovine serum albumin (BSA), brefeldin A from penicillium , complete Freund’s adjuvant (CFA) containing mycobacterium tuberculosis H37RA (10 mg/ml), concanavalin A, dimethyl sulfoxide (DMSO), chondroitinase ABC, cysteine hydrochloric acid (HCL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), eriochrome cyanine, Dulbecco’s Modified Eagle’s Medium (DMEM), 10% buffered formalin, Griess reagent for nitrite, ketoprofen, L-α-Lysophosphatidylcholine (lysolecithin) from egg yolk, phosphate buffered saline (PBS; pH 7.4), pertussis toxin from Bordetella pertussis, Roswell Park Memorial Institute 1640 medium (RPMI), heparinase-III, ionomycin, bacterial lipopolysaccharide (LPS) from E. coli 0111:B4, papain from papaya latex, phorbol 12-myristate 13-acetate (PMA), sodium azide (NaN3 ), sodium citrate, sodium nitrite (NaNO2 ), sodium phosphate (NaH2 PO4 ), SIGMA FAST ® protease inhibitor cocktail tablets, surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide hydrate), and Triton-X-100 were obtained from Sigma-Aldrich Canada (Oakville, ON). .. Fetal bovine serum (FBS), 10,000 U/ml penicillin/10,000 μg/mL streptomycin solution, 200 mM L-glutamine, 1 M 4-(2-hydroyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, and 0.4% trypan blue dye solution were obtained from Invitrogen Canada (Oakville, ON).

    Article Title: Extracellular Vesicles Released by Oxidatively Injured or Intact C2C12 Myotubes Promote Distinct Responses Converging toward Myogenesis
    Article Snippet: .. The protein pellet was dissolved in ISOT buffer (8 M urea, 4% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate), 65 mM DTE (1,4-Dithioerythritol), 40 mM Tris base and added with SIGMAFAST™ Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy)) and sonicated for 5 s on ice. ..

    Radio Ligand Binding Assay:

    Article Title: Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor
    Article Snippet: Total solublized protein of Sf 9 membrane preparations (see above) used in radio ligand binding assay was determined by the Bradford method according to the manufacturers’ protocol (BioRad Protein Assay; BioRad, Munich, Germany). .. Aliquots of homogenized membrane preparations, corresponding to 100 μg of protein, were centrifuged at 50,000 g at 4 °C for 15 min, and the pellets were re-suspended in 50 mM Tris, pH 7.4, supplemented with 1 mM EDTA and protease inhibitors (SIGMAFAST Protease Inhibitor cocktail tablets, Sigma) at a protein concentration of 1,600 μg ml−1 .

    Protein Concentration:

    Article Title: Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor
    Article Snippet: .. Aliquots of homogenized membrane preparations, corresponding to 100 μg of protein, were centrifuged at 50,000 g at 4 °C for 15 min, and the pellets were re-suspended in 50 mM Tris, pH 7.4, supplemented with 1 mM EDTA and protease inhibitors (SIGMAFAST Protease Inhibitor cocktail tablets, Sigma) at a protein concentration of 1,600 μg ml−1 . .. Membrane homogenates (15 μl) were processed and subjected to immunoblotting as described previously with the following modifications: blotting onto the nitrocellulose membrane was performed at 60 mA for 60 min. Primary antibody ANTI-FLAG M1 from mouse (Sigma, order no. F3040, lot SLBK1592V) was diluted 1:500.

    Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy
    Article Snippet: After washing once with cold 1× PBS, the cell pellet was lysed with RIPA lysis buffer (1× PBS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride, 0.25% sodium deoxycholate, 1% NP-40, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride) with 1% freshly added SigmaFAST™ protease inhibitor cocktail (s8830, Sigma-Aldrich, St Louis, MO, USA). .. The resulting mixture was centrifuged at 14,000× g for 10 minutes at 4°C, and the protein concentration in the supernatant was measured using Bradford reagents (Sigma-Aldrich).

    Article Title: Transcriptomic profiles of aging in purified human immune cells
    Article Snippet: Pellet was resuspended in 100 μl modified 4× Laemmli buffer [ ] (4% SDS, 250 mM Tris HCl, no glycerol, no bromophenol blue, no β-mercaptoethanol) mixed 1:1 with 8 M Urea [ ], with SigmaFAST Protease Inhibitor Cocktail Tablet (Sigma-Aldrich, St. Louis, MO). .. Protein concentration was determined using bicinchoninic acid microplate assay (Thermo Scientific, Rockford, IL).

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich). .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    Sonication:

    Article Title: Disease‐associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria
    Article Snippet: Animals were resuspended in RIPA buffer [10 mM Tris–HCl (pH 7.4), 140 mM NaCl, 1% Triton X‐100, 0.1% sodium deoxycholate and 0.1% SDS, 1 mM EDTA] supplemented with protease inhibitors (Sigma, S8830). .. The animals were lysed by douncing followed by sonication for 30 s, five times (amplitude: 40%; Sonics VibraCell VCX130).

    Article Title: Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa
    Article Snippet: For collection of intestinal lavage (IL) samples, small intestine was removed and carefully filled with 3 mL of PBS containing Sigmafast Protease Inhibitor Cocktail (Sigma, USA) using a syringe. .. Tubes were placed in ice and sonicated for 20 minutes in ultrasonic water bath (Elma Schmidbauer, Germany).

    Article Title: Transcriptomic profiles of aging in purified human immune cells
    Article Snippet: Pellet was resuspended in 100 μl modified 4× Laemmli buffer [ ] (4% SDS, 250 mM Tris HCl, no glycerol, no bromophenol blue, no β-mercaptoethanol) mixed 1:1 with 8 M Urea [ ], with SigmaFAST Protease Inhibitor Cocktail Tablet (Sigma-Aldrich, St. Louis, MO). .. Samples were warmed to 55°C, and sonicated 4 × 30 seconds in a Bioruptor (Diagenode, Denville, NJ).

    Article Title: Extracellular Vesicles Released by Oxidatively Injured or Intact C2C12 Myotubes Promote Distinct Responses Converging toward Myogenesis
    Article Snippet: .. The protein pellet was dissolved in ISOT buffer (8 M urea, 4% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate), 65 mM DTE (1,4-Dithioerythritol), 40 mM Tris base and added with SIGMAFAST™ Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy)) and sonicated for 5 s on ice. ..

    MTT Assay:

    Article Title: Surfen, a proteoglycan binding agent, reduces inflammation but inhibits remyelination in murine models of Multiple Sclerosis
    Article Snippet: .. Acetic acid, ammonium sulphamate (H6 N2 O3 S), bovine serum albumin (BSA), brefeldin A from penicillium , complete Freund’s adjuvant (CFA) containing mycobacterium tuberculosis H37RA (10 mg/ml), concanavalin A, dimethyl sulfoxide (DMSO), chondroitinase ABC, cysteine hydrochloric acid (HCL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), eriochrome cyanine, Dulbecco’s Modified Eagle’s Medium (DMEM), 10% buffered formalin, Griess reagent for nitrite, ketoprofen, L-α-Lysophosphatidylcholine (lysolecithin) from egg yolk, phosphate buffered saline (PBS; pH 7.4), pertussis toxin from Bordetella pertussis, Roswell Park Memorial Institute 1640 medium (RPMI), heparinase-III, ionomycin, bacterial lipopolysaccharide (LPS) from E. coli 0111:B4, papain from papaya latex, phorbol 12-myristate 13-acetate (PMA), sodium azide (NaN3 ), sodium citrate, sodium nitrite (NaNO2 ), sodium phosphate (NaH2 PO4 ), SIGMA FAST ® protease inhibitor cocktail tablets, surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide hydrate), and Triton-X-100 were obtained from Sigma-Aldrich Canada (Oakville, ON). .. Fetal bovine serum (FBS), 10,000 U/ml penicillin/10,000 μg/mL streptomycin solution, 200 mM L-glutamine, 1 M 4-(2-hydroyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, and 0.4% trypan blue dye solution were obtained from Invitrogen Canada (Oakville, ON).

    Fluorescence:

    Article Title: An essential contractile ring protein controls cell division in Plasmodium falciparum
    Article Snippet: Quantitative western blotting Parasite pellets were purified by lysing RBCs in 0.05% saponin in PBS with protease inhibitors (SigmaFast Protease Inhibitor Cocktail, Sigma) and boiled in Laemmli buffer. .. Membranes were scanned on a Licor Odyssey CLx imager system and quantified using volumetric measurement of fluorescence intensity.

    Purification:

    Article Title: An essential contractile ring protein controls cell division in Plasmodium falciparum
    Article Snippet: .. Quantitative western blotting Parasite pellets were purified by lysing RBCs in 0.05% saponin in PBS with protease inhibitors (SigmaFast Protease Inhibitor Cocktail, Sigma) and boiled in Laemmli buffer. ..

    Protein Extraction:

    Article Title: Disease‐associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria
    Article Snippet: Paragraph title: C. elegans protein extraction and quantification ... Animals were resuspended in RIPA buffer [10 mM Tris–HCl (pH 7.4), 140 mM NaCl, 1% Triton X‐100, 0.1% sodium deoxycholate and 0.1% SDS, 1 mM EDTA] supplemented with protease inhibitors (Sigma, S8830).

    Article Title: Transcriptomic profiles of aging in purified human immune cells
    Article Snippet: Paragraph title: MCL1 and MRPS12 protein extraction and western blotting ... Pellet was resuspended in 100 μl modified 4× Laemmli buffer [ ] (4% SDS, 250 mM Tris HCl, no glycerol, no bromophenol blue, no β-mercaptoethanol) mixed 1:1 with 8 M Urea [ ], with SigmaFAST Protease Inhibitor Cocktail Tablet (Sigma-Aldrich, St. Louis, MO).

    Mouse Assay:

    Article Title: Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa
    Article Snippet: Two weeks after the second immunization, mice were euthanized by CO2 overdose. .. For collection of intestinal lavage (IL) samples, small intestine was removed and carefully filled with 3 mL of PBS containing Sigmafast Protease Inhibitor Cocktail (Sigma, USA) using a syringe.

    SDS Page:

    Article Title: Transcriptomic profiles of aging in purified human immune cells
    Article Snippet: Pellet was resuspended in 100 μl modified 4× Laemmli buffer [ ] (4% SDS, 250 mM Tris HCl, no glycerol, no bromophenol blue, no β-mercaptoethanol) mixed 1:1 with 8 M Urea [ ], with SigmaFAST Protease Inhibitor Cocktail Tablet (Sigma-Aldrich, St. Louis, MO). .. Samples were mixed 1:4 with 5x Loading Buffer Supplement (50% glycerol, 0.02% bromphenol blue, 12.5% β-mercaptoethanol), separated by SDS-PAGE on NuPage Novex 4-12% Bis-Tris Midi gels (Life Technologies, Grand Island, NY), and transferred to Immobilon Fl (Millipore, Billerica, MA) PVDF membranes.

    Software:

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: Apoptotic cells were determined by analyzing 60,000 ungated cells by using a flow cytometer (Coulter Cytoflex, Beckman, CA, USA) and the FlowJo software. .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    In Vitro:

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: Paragraph title: Cell cultures &in vitro testing ... After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich).

    Lysis:

    Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy
    Article Snippet: .. After washing once with cold 1× PBS, the cell pellet was lysed with RIPA lysis buffer (1× PBS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride, 0.25% sodium deoxycholate, 1% NP-40, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride) with 1% freshly added SigmaFAST™ protease inhibitor cocktail (s8830, Sigma-Aldrich, St Louis, MO, USA). .. The resulting mixture was centrifuged at 14,000× g for 10 minutes at 4°C, and the protein concentration in the supernatant was measured using Bradford reagents (Sigma-Aldrich).

    Article Title: Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment
    Article Snippet: .. After 72 h of treatment, cells were centrifuged at 2500 rpm for 7 min, washed with PBS and finally obtained pellets were incubated with RIPA lysis buffer (Sigma-Aldrich) containing serine/threonine and tyrosine phosphatase inhibitors and a protease inhibitor cocktail 1× (S8830, Sigma-Aldrich). ..

    Article Title: Extracellular Vesicles Released by Oxidatively Injured or Intact C2C12 Myotubes Promote Distinct Responses Converging toward Myogenesis
    Article Snippet: Western Blotting Analysis Protein extracts were obtained from the organic phase after Qiazol cell lysis following Qiagen User Protocol RY16 May-04. .. The protein pellet was dissolved in ISOT buffer (8 M urea, 4% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate), 65 mM DTE (1,4-Dithioerythritol), 40 mM Tris base and added with SIGMAFAST™ Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy)) and sonicated for 5 s on ice.

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  • 93
    Millipore parg inhibitor adp hpd
    PTEN is <t>ADP-ribosylated</t> by tankyrases in vitro and in vivo. ( A , B ) PTEN is PARylated in vivo. ( A ) HCT116 cells were lysed with NETN buffer containing <t>PARG</t> inhibitor <t>ADP-HPD</t> (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PAR antibodies and immunoblotted using the indicated antibodies. ( B ) HCT116 cells were lysed with NETN denaturing buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PTEN antibodies followed by Western blotting as indicated. ( C ) Ribosylation of PTEN by TNKS1 and TNKS2 in vitro. Recombinant TNKS1, TNKS2, and PTEN were subjected to in vitro ribosylation assays in the absence or presence of biotin-labeled NAD + . The recombinant proteins were detected by the indicated antibodies, and the ribosylated proteins were determined with anti-biotin antibody. ( D ) The ribosylation of PTEN by TNKS1 is diminished by tankyrase inhibitor XAV939. The recombinant MBP-PTEN and TNKS1 were subjected to an in vitro ribosylation reaction as described above in the absence or presence of the indicated concentrations of XAV939. ( E ) The catalytic activity of TNKS1 is required for PTEN ribosylation. The MBP-PTEN and immunoprecipitated SFB-tagged wild-type or the catalytically inactive mutant of TNKS1 (TNKS1-PD) were subjected to an in vitro ribosylation reaction followed by Western blotting as indicated. ( F ) The tankyrase-binding motif of PTEN is required for its ribosylation by TNKS1. The recombinant MBP-PTEN, MBP-PTEN-AA, and TNKS1 were subjected to an in vitro ribosylation assay and analyzed by Western blotting as indicated. ( G ) Only double knockdown of TNKS1/2 diminishes the ribosylation of PTEN in vivo. HCT116-derived cells with stable knockdown of TNKS1, TNKS2, TNKS1/2, or PTEN were collected and immunoprecipitated using anti-PTEN antibody. The input and immunoprecipitated proteins were analyzed by Western blotting using the indicated antibodies, and the ribosylation of endogenous PTEN was detected by anti-PAR antibody.
    Parg Inhibitor Adp Hpd, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dmso
    <t>TBZ</t> inhibits angiogenesis in vivo in Xenopus embryos. Formation of Xenopus embryo veins is disrupted, marked by expression of vascular reporter genes (A, B) erg and (C, D) aplnr , contrasting treatment with 1% <t>DMSO</t> control only (A, C) with 1% DMSO, 250 µM TBZ, treated at stage 31 and imaged at stages 35–36 (B, D). PCV, posterior cardinal vein; ISV, intersomitic vein; VV, vitellin veins. Similarly, TBZ disrupts vasculature imaged within a living Xenopus embryo and visualized by vascular specific GFP in kdr:GFP frogs from [19] , contrasting the vasculature of stage 46 animals treated from stage 41 with the 1% DMSO control (E) or 1% DMSO, 250 µM TBZ (F). Scale bar, 200 µm.
    Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore caspase 1
    Inflammasome-unrelated <t>caspase-1</t> activation contributes to A2058 cell apoptosis. ( a ) Serial sections of xenografts as in Figure 1b were stained by indirect immunofluorescence with antibodies to H11/HspB8 (Alexafluor 488-conjugated secondary antibody; green) and caspase-1p20 (Casp1) (AlexaFluor 594-conjugated secondary antibody; red). Double-positive (yellow) cells were counted and the % was calculated relative to the total cell number (determined by DAPI staining, blue). Results are expressed as % positive cells±S.E.M. ( b ) Protein extracts from stably transfected A2058 and A375 cells untreated or treated with Dox (5 μ g/ml; 3 days) in the presence or absence of the caspase-1-specific inhibitor YVAD-CHO (20 μ M) were immunoblotted with antibody to caspase-1p20. Blots were stripped and re-probed with actin antibody. ( c ) Duplicates of the YVAD-CHO-treated cultures in ( b ) were assayed for apoptosis by TUNEL. The cells were counted in five randomly selected 3 mm 2 fields (250 cells each) and the % positive cells calculated relative to the total cell number. Data are expressed as % TUNEL inhibition±S.D. calculated from the formula [1−YVAD-CHO-treated cells/untreated cells)] × 100. ( d ) Extracts from A375 and A2058 xenografts as in Figure 1b (tumors) and from stably transfected A375 and A2058 cells (cell lines) treated or not with Dox (5 μ g/ml; 3 days) were immunoblotted with IL-1 β antibody, stripped and re-probed with actin antibody. Similar results were obtained for IL-18 antibody and by ELISA
    Caspase 1, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore glutathionylation assay thoraces
    DmGSTO1 partially restored mitochondrial F 1 F 0 -ATP synthase activity in park 1 mutants. A , in the presence of GSH, recombinant ATP synthase β subunit was glutathionylated by DmGSTO1A in a dose-dependent manner. B , glutathionylated proteins were immunoprecipitated from thorax extracts with an anti-GSH antibody and were immunoblotted with an anti-ATP synthase β antibody. <t>Glutathionylation</t> of endogenous ATP synthase β subunit in park 1 mutants was regulated by the GSH-conjugating catalytic activity of DmGSTO1A but not by DmGSTO1B. The endogenous levels of the glutathionylated form of the ATP synthase β subunit were decreased even more in park 1 /DmGSTO1 null double mutants. Error bars , S.D. The experimental significance was determined by one-way ANOVA (*, p
    Glutathionylation Assay Thoraces, supplied by Millipore, 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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    Image Search Results


    PTEN is ADP-ribosylated by tankyrases in vitro and in vivo. ( A , B ) PTEN is PARylated in vivo. ( A ) HCT116 cells were lysed with NETN buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PAR antibodies and immunoblotted using the indicated antibodies. ( B ) HCT116 cells were lysed with NETN denaturing buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PTEN antibodies followed by Western blotting as indicated. ( C ) Ribosylation of PTEN by TNKS1 and TNKS2 in vitro. Recombinant TNKS1, TNKS2, and PTEN were subjected to in vitro ribosylation assays in the absence or presence of biotin-labeled NAD + . The recombinant proteins were detected by the indicated antibodies, and the ribosylated proteins were determined with anti-biotin antibody. ( D ) The ribosylation of PTEN by TNKS1 is diminished by tankyrase inhibitor XAV939. The recombinant MBP-PTEN and TNKS1 were subjected to an in vitro ribosylation reaction as described above in the absence or presence of the indicated concentrations of XAV939. ( E ) The catalytic activity of TNKS1 is required for PTEN ribosylation. The MBP-PTEN and immunoprecipitated SFB-tagged wild-type or the catalytically inactive mutant of TNKS1 (TNKS1-PD) were subjected to an in vitro ribosylation reaction followed by Western blotting as indicated. ( F ) The tankyrase-binding motif of PTEN is required for its ribosylation by TNKS1. The recombinant MBP-PTEN, MBP-PTEN-AA, and TNKS1 were subjected to an in vitro ribosylation assay and analyzed by Western blotting as indicated. ( G ) Only double knockdown of TNKS1/2 diminishes the ribosylation of PTEN in vivo. HCT116-derived cells with stable knockdown of TNKS1, TNKS2, TNKS1/2, or PTEN were collected and immunoprecipitated using anti-PTEN antibody. The input and immunoprecipitated proteins were analyzed by Western blotting using the indicated antibodies, and the ribosylation of endogenous PTEN was detected by anti-PAR antibody.

    Journal: Genes & Development

    Article Title: Poly-ADP ribosylation of PTEN by tankyrases promotes PTEN degradation and tumor growth

    doi: 10.1101/gad.251785.114

    Figure Lengend Snippet: PTEN is ADP-ribosylated by tankyrases in vitro and in vivo. ( A , B ) PTEN is PARylated in vivo. ( A ) HCT116 cells were lysed with NETN buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PAR antibodies and immunoblotted using the indicated antibodies. ( B ) HCT116 cells were lysed with NETN denaturing buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PTEN antibodies followed by Western blotting as indicated. ( C ) Ribosylation of PTEN by TNKS1 and TNKS2 in vitro. Recombinant TNKS1, TNKS2, and PTEN were subjected to in vitro ribosylation assays in the absence or presence of biotin-labeled NAD + . The recombinant proteins were detected by the indicated antibodies, and the ribosylated proteins were determined with anti-biotin antibody. ( D ) The ribosylation of PTEN by TNKS1 is diminished by tankyrase inhibitor XAV939. The recombinant MBP-PTEN and TNKS1 were subjected to an in vitro ribosylation reaction as described above in the absence or presence of the indicated concentrations of XAV939. ( E ) The catalytic activity of TNKS1 is required for PTEN ribosylation. The MBP-PTEN and immunoprecipitated SFB-tagged wild-type or the catalytically inactive mutant of TNKS1 (TNKS1-PD) were subjected to an in vitro ribosylation reaction followed by Western blotting as indicated. ( F ) The tankyrase-binding motif of PTEN is required for its ribosylation by TNKS1. The recombinant MBP-PTEN, MBP-PTEN-AA, and TNKS1 were subjected to an in vitro ribosylation assay and analyzed by Western blotting as indicated. ( G ) Only double knockdown of TNKS1/2 diminishes the ribosylation of PTEN in vivo. HCT116-derived cells with stable knockdown of TNKS1, TNKS2, TNKS1/2, or PTEN were collected and immunoprecipitated using anti-PTEN antibody. The input and immunoprecipitated proteins were analyzed by Western blotting using the indicated antibodies, and the ribosylation of endogenous PTEN was detected by anti-PAR antibody.

    Article Snippet: Cycloheximide, MG132, Wiki4, puromycin, G418, and doxycycline were purchased from Sigma-Aldrich; PAPR1/2 inhibitor Olaparib was from LC Laboratories; PARG inhibitor ADP-HPD and JW55 were from Millipore; and XAV939 was from Sigma-Aldrich and Selleckchem.

    Techniques: In Vitro, In Vivo, Protease Inhibitor, Immunoprecipitation, Western Blot, Recombinant, Labeling, Activity Assay, Mutagenesis, Binding Assay, Derivative Assay

    TBZ inhibits angiogenesis in vivo in Xenopus embryos. Formation of Xenopus embryo veins is disrupted, marked by expression of vascular reporter genes (A, B) erg and (C, D) aplnr , contrasting treatment with 1% DMSO control only (A, C) with 1% DMSO, 250 µM TBZ, treated at stage 31 and imaged at stages 35–36 (B, D). PCV, posterior cardinal vein; ISV, intersomitic vein; VV, vitellin veins. Similarly, TBZ disrupts vasculature imaged within a living Xenopus embryo and visualized by vascular specific GFP in kdr:GFP frogs from [19] , contrasting the vasculature of stage 46 animals treated from stage 41 with the 1% DMSO control (E) or 1% DMSO, 250 µM TBZ (F). Scale bar, 200 µm.

    Journal: PLoS Biology

    Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation

    doi: 10.1371/journal.pbio.1001379

    Figure Lengend Snippet: TBZ inhibits angiogenesis in vivo in Xenopus embryos. Formation of Xenopus embryo veins is disrupted, marked by expression of vascular reporter genes (A, B) erg and (C, D) aplnr , contrasting treatment with 1% DMSO control only (A, C) with 1% DMSO, 250 µM TBZ, treated at stage 31 and imaged at stages 35–36 (B, D). PCV, posterior cardinal vein; ISV, intersomitic vein; VV, vitellin veins. Similarly, TBZ disrupts vasculature imaged within a living Xenopus embryo and visualized by vascular specific GFP in kdr:GFP frogs from [19] , contrasting the vasculature of stage 46 animals treated from stage 41 with the 1% DMSO control (E) or 1% DMSO, 250 µM TBZ (F). Scale bar, 200 µm.

    Article Snippet: Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem).

    Techniques: In Vivo, Expressing

    TBZ significantly disrupts tube formation in cultured human umbilical vein endothelial cells (HUVECs), an in vitro capillary model. Here, we show effects of 1% DMSO-treated control (A) versus 1% DMSO, 100 µM TBZ (B) and 1% DMSO, 250 µM TBZ (C). Scale bar, 100 µm. (D) Tube disruption is dose-dependent and comparable to that from silencing known pro-angiogenic gene HOXA9 .

    Journal: PLoS Biology

    Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation

    doi: 10.1371/journal.pbio.1001379

    Figure Lengend Snippet: TBZ significantly disrupts tube formation in cultured human umbilical vein endothelial cells (HUVECs), an in vitro capillary model. Here, we show effects of 1% DMSO-treated control (A) versus 1% DMSO, 100 µM TBZ (B) and 1% DMSO, 250 µM TBZ (C). Scale bar, 100 µm. (D) Tube disruption is dose-dependent and comparable to that from silencing known pro-angiogenic gene HOXA9 .

    Article Snippet: Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem).

    Techniques: Cell Culture, In Vitro

    TBZ impedes migration of HUVECs in a wound scratch assay, but treatment with the Rho Kinase inhibitor Y27632 reverses TBZ's effects. (A) The effects of 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ, and 1% DMSO, 250 µM TBZ, 10 µM Y27632. Scale bar, 200 µm. (B) quantifies the dose-dependent suppression of TBZ inhibition by Y27632. Error bars represent the mean ± 1 s.d. across 3 wells (1 of 3 trials). TBZ results in disorganization of actin stress fibers, as shown in (C) for 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ-treated cells. Scale bar, 20 µm.

    Journal: PLoS Biology

    Article Title: Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting AgentEvolution-Based Drug Discovery: Antifungal Also Disrupts Blood Vessel Formation

    doi: 10.1371/journal.pbio.1001379

    Figure Lengend Snippet: TBZ impedes migration of HUVECs in a wound scratch assay, but treatment with the Rho Kinase inhibitor Y27632 reverses TBZ's effects. (A) The effects of 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ, and 1% DMSO, 250 µM TBZ, 10 µM Y27632. Scale bar, 200 µm. (B) quantifies the dose-dependent suppression of TBZ inhibition by Y27632. Error bars represent the mean ± 1 s.d. across 3 wells (1 of 3 trials). TBZ results in disorganization of actin stress fibers, as shown in (C) for 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ-treated cells. Scale bar, 20 µm.

    Article Snippet: Mass Spectrometry HUVECs were treated with 1% DMSO or 1% DMSO, 250 µM TBZ for 24 h, and lysed by Dounce homogenization in low salt buffer (10 mM Tris-HCl, pH 8.8, 10 mM KCl, 1.5 mM MgCl2 ) with 0.5 mM DTT and protease inhibitor mixture (Calbiochem).

    Techniques: Migration, Wound Healing Assay, Inhibition

    Inflammasome-unrelated caspase-1 activation contributes to A2058 cell apoptosis. ( a ) Serial sections of xenografts as in Figure 1b were stained by indirect immunofluorescence with antibodies to H11/HspB8 (Alexafluor 488-conjugated secondary antibody; green) and caspase-1p20 (Casp1) (AlexaFluor 594-conjugated secondary antibody; red). Double-positive (yellow) cells were counted and the % was calculated relative to the total cell number (determined by DAPI staining, blue). Results are expressed as % positive cells±S.E.M. ( b ) Protein extracts from stably transfected A2058 and A375 cells untreated or treated with Dox (5 μ g/ml; 3 days) in the presence or absence of the caspase-1-specific inhibitor YVAD-CHO (20 μ M) were immunoblotted with antibody to caspase-1p20. Blots were stripped and re-probed with actin antibody. ( c ) Duplicates of the YVAD-CHO-treated cultures in ( b ) were assayed for apoptosis by TUNEL. The cells were counted in five randomly selected 3 mm 2 fields (250 cells each) and the % positive cells calculated relative to the total cell number. Data are expressed as % TUNEL inhibition±S.D. calculated from the formula [1−YVAD-CHO-treated cells/untreated cells)] × 100. ( d ) Extracts from A375 and A2058 xenografts as in Figure 1b (tumors) and from stably transfected A375 and A2058 cells (cell lines) treated or not with Dox (5 μ g/ml; 3 days) were immunoblotted with IL-1 β antibody, stripped and re-probed with actin antibody. Similar results were obtained for IL-18 antibody and by ELISA

    Journal: Cell Death & Disease

    Article Title: Restored expression of the atypical heat shock protein H11/HspB8 inhibits the growth of genetically diverse melanoma tumors through activation of novel TAK1-dependent death pathways

    doi: 10.1038/cddis.2012.108

    Figure Lengend Snippet: Inflammasome-unrelated caspase-1 activation contributes to A2058 cell apoptosis. ( a ) Serial sections of xenografts as in Figure 1b were stained by indirect immunofluorescence with antibodies to H11/HspB8 (Alexafluor 488-conjugated secondary antibody; green) and caspase-1p20 (Casp1) (AlexaFluor 594-conjugated secondary antibody; red). Double-positive (yellow) cells were counted and the % was calculated relative to the total cell number (determined by DAPI staining, blue). Results are expressed as % positive cells±S.E.M. ( b ) Protein extracts from stably transfected A2058 and A375 cells untreated or treated with Dox (5 μ g/ml; 3 days) in the presence or absence of the caspase-1-specific inhibitor YVAD-CHO (20 μ M) were immunoblotted with antibody to caspase-1p20. Blots were stripped and re-probed with actin antibody. ( c ) Duplicates of the YVAD-CHO-treated cultures in ( b ) were assayed for apoptosis by TUNEL. The cells were counted in five randomly selected 3 mm 2 fields (250 cells each) and the % positive cells calculated relative to the total cell number. Data are expressed as % TUNEL inhibition±S.D. calculated from the formula [1−YVAD-CHO-treated cells/untreated cells)] × 100. ( d ) Extracts from A375 and A2058 xenografts as in Figure 1b (tumors) and from stably transfected A375 and A2058 cells (cell lines) treated or not with Dox (5 μ g/ml; 3 days) were immunoblotted with IL-1 β antibody, stripped and re-probed with actin antibody. Similar results were obtained for IL-18 antibody and by ELISA

    Article Snippet: The inhibitors specific for caspase-1 (YVAD-CHO, cell permeable), p38MAPK (SB203580) and caspase-3 (Z-DQMD-fmk) were from Calbiochem (La Jolla, CA, USA) and the pancaspase inhibitor zVAD-fmk from Promega (Madison, WI, USA).

    Techniques: Activation Assay, Staining, Immunofluorescence, Stable Transfection, Transfection, TUNEL Assay, Inhibition, Enzyme-linked Immunosorbent Assay

    Caspase-1 activation is through TAK1-dependent ASC upregulation. ( a ) Protein extracts from stably transfected A375 cells untreated or treated with Dox (5 μ g/ml; 3 days) in the absence or presence of the TAK1 dominant-negative mutant K63W or the empty vector were immunoblotted with antibodies to activated caspase-7 (casp7p20), caspase-3 (casp3p20) or actin. Blots were stripped between probings. ( b ) Stably transfected A2058 cells as in ( a ) were stained with antibody to caspase-1p20 (AlexaFluor 488-conjugated secondary antibody; green) and DAPI (total cell number; blue). ( c ) Results for Dox-treated cells in ( a ) (densitometric units) and ( b ) (% positive cells) were averaged for three independent experiments and the % inhibition calculated from the formula [1−(K63W-transfected cells/untransfected cells)] × 100. ( d ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml; 3 days) in the presence or absence of the p38MAPK-specific inhibitor SB203580 (10 μ M) were immunoblotted with casp3p20, antibody, sequentially stripped and re-probed with antibodies to activated caspase-1 (casp1p20) and actin. ( e ) Extracts from stably transfected A375 and A2058 cells treated as in ( a ) were immunoblotted with antibodies to ASC or actin. Data were quantified by densitometric scanning and the results are expressed as ASC/actin densitometric units±S.D. ( f ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml; 3 days) were immunoprecipitated (IP) with ASC antibody or preimmune IgG and immunoblotted with antibodies to caspase 1 (recognizes the pro- and activated (p20) forms) and ASC. ( g ) Extracts from stably transfected A2058 cells given Dox (5 μ g/ml; 3 days) in the absence or presence of ASC aODN or sODN (30 μ M) were immunoblotted with antibodies to caspase1p20, ASC and actin. Data were quantified by densitometric scanning and the results are expressed as ASC or Casp1/actin densitometric units±S.D.

    Journal: Cell Death & Disease

    Article Title: Restored expression of the atypical heat shock protein H11/HspB8 inhibits the growth of genetically diverse melanoma tumors through activation of novel TAK1-dependent death pathways

    doi: 10.1038/cddis.2012.108

    Figure Lengend Snippet: Caspase-1 activation is through TAK1-dependent ASC upregulation. ( a ) Protein extracts from stably transfected A375 cells untreated or treated with Dox (5 μ g/ml; 3 days) in the absence or presence of the TAK1 dominant-negative mutant K63W or the empty vector were immunoblotted with antibodies to activated caspase-7 (casp7p20), caspase-3 (casp3p20) or actin. Blots were stripped between probings. ( b ) Stably transfected A2058 cells as in ( a ) were stained with antibody to caspase-1p20 (AlexaFluor 488-conjugated secondary antibody; green) and DAPI (total cell number; blue). ( c ) Results for Dox-treated cells in ( a ) (densitometric units) and ( b ) (% positive cells) were averaged for three independent experiments and the % inhibition calculated from the formula [1−(K63W-transfected cells/untransfected cells)] × 100. ( d ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml; 3 days) in the presence or absence of the p38MAPK-specific inhibitor SB203580 (10 μ M) were immunoblotted with casp3p20, antibody, sequentially stripped and re-probed with antibodies to activated caspase-1 (casp1p20) and actin. ( e ) Extracts from stably transfected A375 and A2058 cells treated as in ( a ) were immunoblotted with antibodies to ASC or actin. Data were quantified by densitometric scanning and the results are expressed as ASC/actin densitometric units±S.D. ( f ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml; 3 days) were immunoprecipitated (IP) with ASC antibody or preimmune IgG and immunoblotted with antibodies to caspase 1 (recognizes the pro- and activated (p20) forms) and ASC. ( g ) Extracts from stably transfected A2058 cells given Dox (5 μ g/ml; 3 days) in the absence or presence of ASC aODN or sODN (30 μ M) were immunoblotted with antibodies to caspase1p20, ASC and actin. Data were quantified by densitometric scanning and the results are expressed as ASC or Casp1/actin densitometric units±S.D.

    Article Snippet: The inhibitors specific for caspase-1 (YVAD-CHO, cell permeable), p38MAPK (SB203580) and caspase-3 (Z-DQMD-fmk) were from Calbiochem (La Jolla, CA, USA) and the pancaspase inhibitor zVAD-fmk from Promega (Madison, WI, USA).

    Techniques: Activation Assay, Stable Transfection, Transfection, Dominant Negative Mutation, Plasmid Preparation, Staining, Inhibition, Immunoprecipitation

    Caspase-1 cleaves Beclin-1 and induces apoptosis in A2058 cells. ( a ) Protein extracts from A375 and A2058 xenografts as in Figure 1b were immunoblotted with antibodies to Beclin-1, followed by actin. Molecular weights are shown on the right. ( b ) Serial sections of A2058 and A375 xenografts as in Figure 1b were stained with antibodies to Beclin-1 (AlexaFluor 488-conjugated secondary antibody; green) and CoxIV (mitochondrial marker; AlexaFluor 594-conjugated secondary antibody; red). The cells with CoxIV/Beclin-1 co-localization (yellow) were counted and the % calculated relative to the total cell number (determined by DAPI staining, blue). ( c ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml) in the presence or absence of the caspase-1 inhibitor YVAD-CHO (20 μ M), were immunoblotted with antibodies to Beclin-1 or actin and the data quantified and expressed as in Figure 6

    Journal: Cell Death & Disease

    Article Title: Restored expression of the atypical heat shock protein H11/HspB8 inhibits the growth of genetically diverse melanoma tumors through activation of novel TAK1-dependent death pathways

    doi: 10.1038/cddis.2012.108

    Figure Lengend Snippet: Caspase-1 cleaves Beclin-1 and induces apoptosis in A2058 cells. ( a ) Protein extracts from A375 and A2058 xenografts as in Figure 1b were immunoblotted with antibodies to Beclin-1, followed by actin. Molecular weights are shown on the right. ( b ) Serial sections of A2058 and A375 xenografts as in Figure 1b were stained with antibodies to Beclin-1 (AlexaFluor 488-conjugated secondary antibody; green) and CoxIV (mitochondrial marker; AlexaFluor 594-conjugated secondary antibody; red). The cells with CoxIV/Beclin-1 co-localization (yellow) were counted and the % calculated relative to the total cell number (determined by DAPI staining, blue). ( c ) Extracts from stably transfected A2058 cells treated or not with Dox (5 μ g/ml) in the presence or absence of the caspase-1 inhibitor YVAD-CHO (20 μ M), were immunoblotted with antibodies to Beclin-1 or actin and the data quantified and expressed as in Figure 6

    Article Snippet: The inhibitors specific for caspase-1 (YVAD-CHO, cell permeable), p38MAPK (SB203580) and caspase-3 (Z-DQMD-fmk) were from Calbiochem (La Jolla, CA, USA) and the pancaspase inhibitor zVAD-fmk from Promega (Madison, WI, USA).

    Techniques: Staining, Marker, Stable Transfection, Transfection

    Schematic representation of the H11/HspB8-induced death pathways in A2058 and A375 cells. H11/HspB8 activates TAK1 in both melanoma lines. In A375 cells, the TAK1/p38MAPK pathway activates caspase-3/7 to cause apoptosis. In A2058 cells, the TAK1/p38MAPK pathway activates caspase-3, but TAK1 also activates caspase-1 through ASC upregulation and upregulates Beclin-1 through mTOR phosphorylation at S2481 (pmTORS2481). Caspase-1 cleaves Beclin-1 to promote apoptosis, but Beclin-1 also contributes to cell death through still unknown tumor-suppressor functions

    Journal: Cell Death & Disease

    Article Title: Restored expression of the atypical heat shock protein H11/HspB8 inhibits the growth of genetically diverse melanoma tumors through activation of novel TAK1-dependent death pathways

    doi: 10.1038/cddis.2012.108

    Figure Lengend Snippet: Schematic representation of the H11/HspB8-induced death pathways in A2058 and A375 cells. H11/HspB8 activates TAK1 in both melanoma lines. In A375 cells, the TAK1/p38MAPK pathway activates caspase-3/7 to cause apoptosis. In A2058 cells, the TAK1/p38MAPK pathway activates caspase-3, but TAK1 also activates caspase-1 through ASC upregulation and upregulates Beclin-1 through mTOR phosphorylation at S2481 (pmTORS2481). Caspase-1 cleaves Beclin-1 to promote apoptosis, but Beclin-1 also contributes to cell death through still unknown tumor-suppressor functions

    Article Snippet: The inhibitors specific for caspase-1 (YVAD-CHO, cell permeable), p38MAPK (SB203580) and caspase-3 (Z-DQMD-fmk) were from Calbiochem (La Jolla, CA, USA) and the pancaspase inhibitor zVAD-fmk from Promega (Madison, WI, USA).

    Techniques:

    DmGSTO1 partially restored mitochondrial F 1 F 0 -ATP synthase activity in park 1 mutants. A , in the presence of GSH, recombinant ATP synthase β subunit was glutathionylated by DmGSTO1A in a dose-dependent manner. B , glutathionylated proteins were immunoprecipitated from thorax extracts with an anti-GSH antibody and were immunoblotted with an anti-ATP synthase β antibody. Glutathionylation of endogenous ATP synthase β subunit in park 1 mutants was regulated by the GSH-conjugating catalytic activity of DmGSTO1A but not by DmGSTO1B. The endogenous levels of the glutathionylated form of the ATP synthase β subunit were decreased even more in park 1 /DmGSTO1 null double mutants. Error bars , S.D. The experimental significance was determined by one-way ANOVA (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Glutathione S-Transferase Omega 1 Activity Is Sufficient to Suppress Neurodegeneration in a Drosophila Model of Parkinson Disease *

    doi: 10.1074/jbc.M111.291179

    Figure Lengend Snippet: DmGSTO1 partially restored mitochondrial F 1 F 0 -ATP synthase activity in park 1 mutants. A , in the presence of GSH, recombinant ATP synthase β subunit was glutathionylated by DmGSTO1A in a dose-dependent manner. B , glutathionylated proteins were immunoprecipitated from thorax extracts with an anti-GSH antibody and were immunoblotted with an anti-ATP synthase β antibody. Glutathionylation of endogenous ATP synthase β subunit in park 1 mutants was regulated by the GSH-conjugating catalytic activity of DmGSTO1A but not by DmGSTO1B. The endogenous levels of the glutathionylated form of the ATP synthase β subunit were decreased even more in park 1 /DmGSTO1 null double mutants. Error bars , S.D. The experimental significance was determined by one-way ANOVA (*, p

    Article Snippet: Immunoprecipitation and Glutathionylation Assay Thoraces from 3-day-old male flies were homogenized in lysis buffer containing 1× protease inhibitor mixture (Calbiochem-Merck4Biosciences).

    Techniques: Activity Assay, Recombinant, Immunoprecipitation