protease inhibitor  (Millipore)

 
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    • 97
    Name:
    Protease Inhibitor Cocktail
    Description:

    Catalog Number:
    P8340
    Price:
    None
    Applications:
    Protease Inhibitor Cocktail has been used-. as a buffer component during sonication of GFP (green fluorescent protein)-huntingtin-transfected HEK 293 cells in GST (glutathione S-transferase) pull down assay. as a component of lysis buffer. as a component of radioimmunoprecipitation assay buffer (RIPA)
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    Structured Review

    Millipore protease inhibitor

    https://www.bioz.com/result/protease inhibitor/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor - by Bioz Stars, 2021-07
    97/100 stars

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    Related Articles

    Expressing:

    Article Title: Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions
    Article Snippet: In short, proteins were expressed in Sf9 insect cells (Expression Systems, Cat#94-001F) using the baculovirus system and produced as C-terminal EGFP fusions with an N-terminal maltose-binding protein (MBP) tag and a C-terminal hexahistidine (His6 ) tag. .. Cells expressing MBP-FUS-EGFP-His6 and MBP-FUS(G156E)-EGFP-His6 were harvested 72 h post infection by centrifugation at 2000 rpm for 5 min and resuspended in 50 mM Tris-HCl (pH 7.4), 1 M KCl, 5% (w /v ) glycerol, 1 mM DTT, 10 mM imidazole supplemented with EDTA-free protease inhibitor cocktail set III (Calbiochem) and 0.25 U/mL benzonase (in-house, provided by the MPI-CBG protein expression facility). .. Cell lysis was done with an LM20 shear homogenizer (Microfluidics) at 5000 psi.

    Infection:

    Article Title: Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions
    Article Snippet: In short, proteins were expressed in Sf9 insect cells (Expression Systems, Cat#94-001F) using the baculovirus system and produced as C-terminal EGFP fusions with an N-terminal maltose-binding protein (MBP) tag and a C-terminal hexahistidine (His6 ) tag. .. Cells expressing MBP-FUS-EGFP-His6 and MBP-FUS(G156E)-EGFP-His6 were harvested 72 h post infection by centrifugation at 2000 rpm for 5 min and resuspended in 50 mM Tris-HCl (pH 7.4), 1 M KCl, 5% (w /v ) glycerol, 1 mM DTT, 10 mM imidazole supplemented with EDTA-free protease inhibitor cocktail set III (Calbiochem) and 0.25 U/mL benzonase (in-house, provided by the MPI-CBG protein expression facility). .. Cell lysis was done with an LM20 shear homogenizer (Microfluidics) at 5000 psi.

    Centrifugation:

    Article Title: Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions
    Article Snippet: In short, proteins were expressed in Sf9 insect cells (Expression Systems, Cat#94-001F) using the baculovirus system and produced as C-terminal EGFP fusions with an N-terminal maltose-binding protein (MBP) tag and a C-terminal hexahistidine (His6 ) tag. .. Cells expressing MBP-FUS-EGFP-His6 and MBP-FUS(G156E)-EGFP-His6 were harvested 72 h post infection by centrifugation at 2000 rpm for 5 min and resuspended in 50 mM Tris-HCl (pH 7.4), 1 M KCl, 5% (w /v ) glycerol, 1 mM DTT, 10 mM imidazole supplemented with EDTA-free protease inhibitor cocktail set III (Calbiochem) and 0.25 U/mL benzonase (in-house, provided by the MPI-CBG protein expression facility). .. Cell lysis was done with an LM20 shear homogenizer (Microfluidics) at 5000 psi.

    Article Title: Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
    Article Snippet: After adding 1× PBS (GIBCO BRL), cell suspension was centrifuged (10 min, 1,000 rpm) and the pellet washed twice with 1 ml PBS. .. Pellets were homogenized by vigorous pipetting in 1 ml HEK293 lysis buffer (500 mM NaCl, 50 mM NaH2 PO4 , 1% Triton X-100, 1% protease inhibitor cocktail for mammalian tissues [Sigma-Aldrich], pH 8.0) and rotating at 4°C for 1–2 h. To pellet cellular debris and DNA the samples were centrifuged at 20,000 g and 4°C for 60 min. A clear 100-μl supernatant sample was taken and proteins precipitated by adding 400 μl methanol, 100 μl chloroform, and 300 μl water, respectively, and centrifugation at 20,000 g for 3 min at 4°C. .. Finally, the protein pellets were resuspended in 50 μl Laemmli sample buffer (Bio-Rad Laboratories) containing 5% β-mercapto-ethanol (Bio-Rad Laboratories).

    Protease Inhibitor:

    Article Title: Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions
    Article Snippet: In short, proteins were expressed in Sf9 insect cells (Expression Systems, Cat#94-001F) using the baculovirus system and produced as C-terminal EGFP fusions with an N-terminal maltose-binding protein (MBP) tag and a C-terminal hexahistidine (His6 ) tag. .. Cells expressing MBP-FUS-EGFP-His6 and MBP-FUS(G156E)-EGFP-His6 were harvested 72 h post infection by centrifugation at 2000 rpm for 5 min and resuspended in 50 mM Tris-HCl (pH 7.4), 1 M KCl, 5% (w /v ) glycerol, 1 mM DTT, 10 mM imidazole supplemented with EDTA-free protease inhibitor cocktail set III (Calbiochem) and 0.25 U/mL benzonase (in-house, provided by the MPI-CBG protein expression facility). .. Cell lysis was done with an LM20 shear homogenizer (Microfluidics) at 5000 psi.

    Article Title: The Herpes Simplex Virus 1 UL51 Protein Interacts with the UL7 Protein and Plays a Role in Its Recruitment into the Virion
    Article Snippet: pUL51-FLAG was purified from Vero or HEp-2 cells that had been infected with 5 PFU/cell of wild-type or recombinant tagged HSV-1 for 16 h. Infected cell monolayers from 100-mm cultures were washed with 5 ml of phosphate-buffered saline (PBS), and then the cells were scraped into 3 ml of PBS and pelleted at 110 × g for 10 min. .. The cell pellets were resuspended in 1.5 ml coimmunoprecipitation (co-IP) buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1× Sigma protease inhibitor cocktail), transferred to microcentrifuge tubes, and incubated on ice for 3 min. .. Nuclei and other cellular debris were pelleted by centrifugation at 10,000 rpm in a microcentrifuge for 10 min, and the supernatant was transferred to a fresh tube.

    Article Title: Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
    Article Snippet: After adding 1× PBS (GIBCO BRL), cell suspension was centrifuged (10 min, 1,000 rpm) and the pellet washed twice with 1 ml PBS. .. Pellets were homogenized by vigorous pipetting in 1 ml HEK293 lysis buffer (500 mM NaCl, 50 mM NaH2 PO4 , 1% Triton X-100, 1% protease inhibitor cocktail for mammalian tissues [Sigma-Aldrich], pH 8.0) and rotating at 4°C for 1–2 h. To pellet cellular debris and DNA the samples were centrifuged at 20,000 g and 4°C for 60 min. A clear 100-μl supernatant sample was taken and proteins precipitated by adding 400 μl methanol, 100 μl chloroform, and 300 μl water, respectively, and centrifugation at 20,000 g for 3 min at 4°C. .. Finally, the protein pellets were resuspended in 50 μl Laemmli sample buffer (Bio-Rad Laboratories) containing 5% β-mercapto-ethanol (Bio-Rad Laboratories).

    Article Title: Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)
    Article Snippet: .. Twenty millilitres of ice-cold 0.01M MES buffer (pH 5.5) containing 0.2M KCl plus protease inhibitors (1mM phenylmethylsulphonyl fluoride and 5 µl of protease inhibitor cocktail; Sigma) was added to the vial, submerging the plant tissue. ..

    Article Title: Tanshinones that selectively block the collagenase activity of cathepsin K provide a novel class of ectosteric antiresorptive agents for bone) Tanshinones that selectively block the collagenase activity of cathepsin K provide a novel class of ectosteric antiresorptive agents for bone
    Article Snippet: .. Human skin fibroblasts (PromoCell GmBH, Heidelberg, Germany) were seeded in a six‐well plate in the presence or absence of tanshinones or an E64‐containing protease inhibitor cocktail (PIC) (Sigma‐Aldrich, St. Louis, MO, USA) in DMEM containing 10% FBS at 37°C and 5% CO2 as previously described (Panwar et al ., ). .. Western blot detection of TGF‐β1 and β‐actin in fibroblast cell lysates was performed with human anti‐TGF‐β1 primary antibody (G1221, Promega BioSciences, Madison, WI, USA) and anti‐β‐actin antibody (622102, BioLegend, San Diego, CA, USA) (1:1000 dilution) incubated overnight at 4°C.

    Article Title: CBP Activity Mediates Effects of the Histone Deacetylase Inhibitor Butyrate on WNT Activity and Apoptosis in Colon Cancer Cells
    Article Snippet: Coimmunoprecipitation For CBP/p300-beta-catenin coimmunoprecipitations, HCT-116 or SW620 cells were incubated overnight (17.5 hr) with or without ICG-001, and nuclei were isolated with the Nuclei EZ Prep kit (Sigma). .. Nuclear pellets were lysed in SDS-containing buffer , , and 100 μg nuclear protein extract was diluted to 1 mL in coimmunoprecipitation (Co-IP) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, and Sigma protease inhibitor cocktail). .. Two μg of either CBP (sc-369, Santa Cruz Biotechnology) or p300 (sc-584, Santa Cruz Biotechnology) antibody were added to the protein samples, and incubated overnight at 4o C with rotation.

    Article Title: Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites
    Article Snippet: .. Cells were washed three times in PBS and lysed in immunoprecipitation (IP) buffer (20 mM HEPES, pH 7.4, 130 mM NaCl, 0.3% protease inhibitor cocktail (Sigma-Aldrich), 10 mM NaF, phosphatase inhibitors (cocktails 2 and 3, Sigma-Aldrich) and 1% NP-40). .. Samples were centrifuged at 10,000×g for 10 min at 4 °C, and the supernatant was then incubated with HA-Trap magnetic microparticles (ChromoTek) for 1 h at 4 °C.

    Article Title: Anti-carcinoembryonic antigen-related cell adhesion molecule antibody for fluorescence visualization of primary colon cancer and metastases in patient-derived orthotopic xenograft mouse models
    Article Snippet: DAB/hematoxylin stained samples and DAPI/fluorescent stained proteins were analyzed with a Leica DMI4000B microscope (20× objective, 10× eyepiece, total magnification 200×). .. Enzyme-linked immunosorbent assay (ELISA) Indicated transfectants were lysed in RIPA buffer containing protease inhibitor cocktail set III (Calbiochem) and centrifuged at 18,000 rpm for 15 min at 4°C. ..

    Co-Immunoprecipitation Assay:

    Article Title: The Herpes Simplex Virus 1 UL51 Protein Interacts with the UL7 Protein and Plays a Role in Its Recruitment into the Virion
    Article Snippet: pUL51-FLAG was purified from Vero or HEp-2 cells that had been infected with 5 PFU/cell of wild-type or recombinant tagged HSV-1 for 16 h. Infected cell monolayers from 100-mm cultures were washed with 5 ml of phosphate-buffered saline (PBS), and then the cells were scraped into 3 ml of PBS and pelleted at 110 × g for 10 min. .. The cell pellets were resuspended in 1.5 ml coimmunoprecipitation (co-IP) buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1× Sigma protease inhibitor cocktail), transferred to microcentrifuge tubes, and incubated on ice for 3 min. .. Nuclei and other cellular debris were pelleted by centrifugation at 10,000 rpm in a microcentrifuge for 10 min, and the supernatant was transferred to a fresh tube.

    Article Title: CBP Activity Mediates Effects of the Histone Deacetylase Inhibitor Butyrate on WNT Activity and Apoptosis in Colon Cancer Cells
    Article Snippet: Coimmunoprecipitation For CBP/p300-beta-catenin coimmunoprecipitations, HCT-116 or SW620 cells were incubated overnight (17.5 hr) with or without ICG-001, and nuclei were isolated with the Nuclei EZ Prep kit (Sigma). .. Nuclear pellets were lysed in SDS-containing buffer , , and 100 μg nuclear protein extract was diluted to 1 mL in coimmunoprecipitation (Co-IP) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, and Sigma protease inhibitor cocktail). .. Two μg of either CBP (sc-369, Santa Cruz Biotechnology) or p300 (sc-584, Santa Cruz Biotechnology) antibody were added to the protein samples, and incubated overnight at 4o C with rotation.

    Incubation:

    Article Title: The Herpes Simplex Virus 1 UL51 Protein Interacts with the UL7 Protein and Plays a Role in Its Recruitment into the Virion
    Article Snippet: pUL51-FLAG was purified from Vero or HEp-2 cells that had been infected with 5 PFU/cell of wild-type or recombinant tagged HSV-1 for 16 h. Infected cell monolayers from 100-mm cultures were washed with 5 ml of phosphate-buffered saline (PBS), and then the cells were scraped into 3 ml of PBS and pelleted at 110 × g for 10 min. .. The cell pellets were resuspended in 1.5 ml coimmunoprecipitation (co-IP) buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1× Sigma protease inhibitor cocktail), transferred to microcentrifuge tubes, and incubated on ice for 3 min. .. Nuclei and other cellular debris were pelleted by centrifugation at 10,000 rpm in a microcentrifuge for 10 min, and the supernatant was transferred to a fresh tube.

    Lysis:

    Article Title: Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
    Article Snippet: After adding 1× PBS (GIBCO BRL), cell suspension was centrifuged (10 min, 1,000 rpm) and the pellet washed twice with 1 ml PBS. .. Pellets were homogenized by vigorous pipetting in 1 ml HEK293 lysis buffer (500 mM NaCl, 50 mM NaH2 PO4 , 1% Triton X-100, 1% protease inhibitor cocktail for mammalian tissues [Sigma-Aldrich], pH 8.0) and rotating at 4°C for 1–2 h. To pellet cellular debris and DNA the samples were centrifuged at 20,000 g and 4°C for 60 min. A clear 100-μl supernatant sample was taken and proteins precipitated by adding 400 μl methanol, 100 μl chloroform, and 300 μl water, respectively, and centrifugation at 20,000 g for 3 min at 4°C. .. Finally, the protein pellets were resuspended in 50 μl Laemmli sample buffer (Bio-Rad Laboratories) containing 5% β-mercapto-ethanol (Bio-Rad Laboratories).

    Immunoprecipitation:

    Article Title: Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites
    Article Snippet: .. Cells were washed three times in PBS and lysed in immunoprecipitation (IP) buffer (20 mM HEPES, pH 7.4, 130 mM NaCl, 0.3% protease inhibitor cocktail (Sigma-Aldrich), 10 mM NaF, phosphatase inhibitors (cocktails 2 and 3, Sigma-Aldrich) and 1% NP-40). .. Samples were centrifuged at 10,000×g for 10 min at 4 °C, and the supernatant was then incubated with HA-Trap magnetic microparticles (ChromoTek) for 1 h at 4 °C.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Anti-carcinoembryonic antigen-related cell adhesion molecule antibody for fluorescence visualization of primary colon cancer and metastases in patient-derived orthotopic xenograft mouse models
    Article Snippet: DAB/hematoxylin stained samples and DAPI/fluorescent stained proteins were analyzed with a Leica DMI4000B microscope (20× objective, 10× eyepiece, total magnification 200×). .. Enzyme-linked immunosorbent assay (ELISA) Indicated transfectants were lysed in RIPA buffer containing protease inhibitor cocktail set III (Calbiochem) and centrifuged at 18,000 rpm for 15 min at 4°C. ..

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    • calpain inhibitors  (Millipore)
    94
    Millipore calpain inhibitors
    Moderate doses of <t>calpain</t> inhibitors improve vascular re-growth and reduce retinal hypoxia associated with ischemic retinopathy ( A ) Animals were treated with vehicle or calpain inhibitor MDL 28170, beginning with the onset of retinopathy for 5 days (P12–17). Whole mount retina vasculature at P17 was stained with Bandeiraea simplicifolia TRITC-lectin (red, upper panels); note that MDL 28170 at 0.25 mg/kg improved vascular re-growth whereas 0.50 mg/kg did not improve re-growth and instead was inhibitory (see Panel C for dose curve). Whole mount retinas were also stained for hypoxia with Hypoxyprobe™ (green, middle panels). Bottom panels are merged images of the upper two panels; green color corresponds to hypoxia in the absence of neovasculature and yellow color (red + green) corresponds to abnormal vascularized area that is still severely hypoxic. Scale bar, 500 µm. ( B ) Quantification of % avascular area, as determined with Bandeiraea simplicifolia TRITC-lectin staining (Panel “A”, upper panels in red), following treatment with vehicle control or calpain inhibitors MDL 28170, PD150606, and ALLN at optimal doses, - see (C) below (*p
    Calpain Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    biotin protein ligase bira  (Millipore)
    93
    Millipore biotin protein ligase bira
    PPARγ activation induces de novo lipid synthesis in a <t>sumoylation</t> dependent manner (A) Sumoylation of endogenous PPARγ in Calu6. PPARγ immunoprecipitates from cell lysates treated with 30 μM of pioglitazone for 24 hours were subjected to western blot using indicated antibodies. (B, C) Sumoylation of PPARγWT or PPARγSUMO (K107, 395R). (B) HEK293 cells were co-transfected with plasmids expressing <t>BirA,</t> Flag-SUMO1, and BLRP-PPARγ WT or BLRP-PPARγ SUMO, and further treated with 3 μM of pioglitazone for 24 hours. Streptavidine (strep) immunoprecipitates were subjected to western blot using indicated antibodies. (C) Sumoylation of PPARγ in HBEC cells with stable expression of BLRP-PPARγ WT or BLRP-PPARγ SUMO upon tetracycline induction. Streptavidine immunoprecipitates from HBEC cells infected with pAd-BirA were subjected to western blot using indicated antibodies. (D) ORO lipid staining in HBEC cells. HBEC cells under tetracycline (Tet) ON or OFF conditions were treated with pioglitazone (3 μM) for 5 days in media containing 5% CS, followed by ORO staining to detect lipid droplet accumulation. (E) Metabolic profiling of fatty acids upon ligand activation of PPARγ. HBEC cells were treated with pioglitazone (3 μM) for 5 days, followed by free fatty acid extraction and HPLC analysis to profile lipid content. Values are mean ± S.E.M. Statistical analysis was performed using one-way ANOVA (Turkey’s post-tests). * p
    Biotin Protein Ligase Bira, 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
    https://www.bioz.com/result/biotin protein ligase bira/product/Millipore
    Average 93 stars, based on 1 article reviews
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    s methyl methanethiosulfonate  (Millipore)
    95
    Millipore s methyl methanethiosulfonate
    DHHC21 mediates TCR-induced S-acylation of signaling proteins. (A) Overview of the acyl-resin assisted capture (Acyl-RAC) assay. Free cysteine thiols (-SH) are irreversibly blocked by S-methyl <t>methanethiosulfonate</t> (MMTS) following cell lysis. Thioester bonds between cysteines and acyl groups are then specifically cleaved by neutral hydroxylamine (HA). The newly formed free thiol groups are captured by the thiol-reactive sepharose and S-acylated proteins detected by immunoblotting. (B) TCR-induced protein S-acylation. CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with cross-linked anti-CD3/CD28 antibodies for indicated times and protein S-acylation (acyl-) was determined using Acyl-RAC assay. Samples not treated with hydroxylamine (-HA) were used as a negative control. β-Actin, a known S-acylated protein ( 32 ), was used as a loading control.
    S Methyl Methanethiosulfonate, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tbz  (Millipore)
    94
    Millipore tbz
    <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.
    Tbz, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tbz/product/Millipore
    Average 94 stars, based on 1 article reviews
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    tbz - by Bioz Stars, 2021-07
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    Image Search Results


    Moderate doses of calpain inhibitors improve vascular re-growth and reduce retinal hypoxia associated with ischemic retinopathy ( A ) Animals were treated with vehicle or calpain inhibitor MDL 28170, beginning with the onset of retinopathy for 5 days (P12–17). Whole mount retina vasculature at P17 was stained with Bandeiraea simplicifolia TRITC-lectin (red, upper panels); note that MDL 28170 at 0.25 mg/kg improved vascular re-growth whereas 0.50 mg/kg did not improve re-growth and instead was inhibitory (see Panel C for dose curve). Whole mount retinas were also stained for hypoxia with Hypoxyprobe™ (green, middle panels). Bottom panels are merged images of the upper two panels; green color corresponds to hypoxia in the absence of neovasculature and yellow color (red + green) corresponds to abnormal vascularized area that is still severely hypoxic. Scale bar, 500 µm. ( B ) Quantification of % avascular area, as determined with Bandeiraea simplicifolia TRITC-lectin staining (Panel “A”, upper panels in red), following treatment with vehicle control or calpain inhibitors MDL 28170, PD150606, and ALLN at optimal doses, - see (C) below (*p

    Journal: Biochimica et biophysica acta

    Article Title: Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion

    doi: 10.1016/j.bbadis.2010.08.008

    Figure Lengend Snippet: Moderate doses of calpain inhibitors improve vascular re-growth and reduce retinal hypoxia associated with ischemic retinopathy ( A ) Animals were treated with vehicle or calpain inhibitor MDL 28170, beginning with the onset of retinopathy for 5 days (P12–17). Whole mount retina vasculature at P17 was stained with Bandeiraea simplicifolia TRITC-lectin (red, upper panels); note that MDL 28170 at 0.25 mg/kg improved vascular re-growth whereas 0.50 mg/kg did not improve re-growth and instead was inhibitory (see Panel C for dose curve). Whole mount retinas were also stained for hypoxia with Hypoxyprobe™ (green, middle panels). Bottom panels are merged images of the upper two panels; green color corresponds to hypoxia in the absence of neovasculature and yellow color (red + green) corresponds to abnormal vascularized area that is still severely hypoxic. Scale bar, 500 µm. ( B ) Quantification of % avascular area, as determined with Bandeiraea simplicifolia TRITC-lectin staining (Panel “A”, upper panels in red), following treatment with vehicle control or calpain inhibitors MDL 28170, PD150606, and ALLN at optimal doses, - see (C) below (*p

    Article Snippet: Most significantly, we found that hypoxia severely disrupted actin cables in retinal MVECs undergoing capillary morphogenesis and that calpain inhibitors not only restored the actin cytoskeleton during hypoxia but also promoted coordinate alignment of actin cables between neighboring MVECs within vascular network ( ).

    Techniques: Staining

    Hypoxia induces calpain activity and disassembles the actin cytoskeleton in retinal MVECs; moderate calpain inhibition restores actin cables in hypoxia ( A ) Retinal MVECs cultured in normoxia (21 % O 2 ) were subjected to hypoxia (5% O 2 ) for 2 h in the presence or absence of calpain inhibitors (calpastatin 200 nM or MDL 28170, at doses indicated) and assayed for calpain activity as described in methods. **p

    Journal: Biochimica et biophysica acta

    Article Title: Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion

    doi: 10.1016/j.bbadis.2010.08.008

    Figure Lengend Snippet: Hypoxia induces calpain activity and disassembles the actin cytoskeleton in retinal MVECs; moderate calpain inhibition restores actin cables in hypoxia ( A ) Retinal MVECs cultured in normoxia (21 % O 2 ) were subjected to hypoxia (5% O 2 ) for 2 h in the presence or absence of calpain inhibitors (calpastatin 200 nM or MDL 28170, at doses indicated) and assayed for calpain activity as described in methods. **p

    Article Snippet: Most significantly, we found that hypoxia severely disrupted actin cables in retinal MVECs undergoing capillary morphogenesis and that calpain inhibitors not only restored the actin cytoskeleton during hypoxia but also promoted coordinate alignment of actin cables between neighboring MVECs within vascular network ( ).

    Techniques: Activity Assay, Inhibition, Cell Culture

    Calpain inhibitors improve organization and alignment of endothelial cell actin cables during capillary morphogenesis in vitro and neovascularization in vivo ; optimal improvement is achieved with 30–35% inhibition of calpain activity ( A ) F-actin (stained with FITC-phalloidin) in retinal MVECs undergoing capillary morphogenesis in vitro in response to overlay with collagen I. Scale bar, 25 µm. Calpain inhibitor doses: MDL 28170 (10 nM), calpastatin peptide (200 nM). ( B ) Calpain inhibitors improve capillary morphogenesis in vitro as measured by increased cord length, increased formation of inter-connected polygon networks, and reduction in blind ends. ***p

    Journal: Biochimica et biophysica acta

    Article Title: Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion

    doi: 10.1016/j.bbadis.2010.08.008

    Figure Lengend Snippet: Calpain inhibitors improve organization and alignment of endothelial cell actin cables during capillary morphogenesis in vitro and neovascularization in vivo ; optimal improvement is achieved with 30–35% inhibition of calpain activity ( A ) F-actin (stained with FITC-phalloidin) in retinal MVECs undergoing capillary morphogenesis in vitro in response to overlay with collagen I. Scale bar, 25 µm. Calpain inhibitor doses: MDL 28170 (10 nM), calpastatin peptide (200 nM). ( B ) Calpain inhibitors improve capillary morphogenesis in vitro as measured by increased cord length, increased formation of inter-connected polygon networks, and reduction in blind ends. ***p

    Article Snippet: Most significantly, we found that hypoxia severely disrupted actin cables in retinal MVECs undergoing capillary morphogenesis and that calpain inhibitors not only restored the actin cytoskeleton during hypoxia but also promoted coordinate alignment of actin cables between neighboring MVECs within vascular network ( ).

    Techniques: In Vitro, In Vivo, Inhibition, Activity Assay, Staining

    PPARγ activation induces de novo lipid synthesis in a sumoylation dependent manner (A) Sumoylation of endogenous PPARγ in Calu6. PPARγ immunoprecipitates from cell lysates treated with 30 μM of pioglitazone for 24 hours were subjected to western blot using indicated antibodies. (B, C) Sumoylation of PPARγWT or PPARγSUMO (K107, 395R). (B) HEK293 cells were co-transfected with plasmids expressing BirA, Flag-SUMO1, and BLRP-PPARγ WT or BLRP-PPARγ SUMO, and further treated with 3 μM of pioglitazone for 24 hours. Streptavidine (strep) immunoprecipitates were subjected to western blot using indicated antibodies. (C) Sumoylation of PPARγ in HBEC cells with stable expression of BLRP-PPARγ WT or BLRP-PPARγ SUMO upon tetracycline induction. Streptavidine immunoprecipitates from HBEC cells infected with pAd-BirA were subjected to western blot using indicated antibodies. (D) ORO lipid staining in HBEC cells. HBEC cells under tetracycline (Tet) ON or OFF conditions were treated with pioglitazone (3 μM) for 5 days in media containing 5% CS, followed by ORO staining to detect lipid droplet accumulation. (E) Metabolic profiling of fatty acids upon ligand activation of PPARγ. HBEC cells were treated with pioglitazone (3 μM) for 5 days, followed by free fatty acid extraction and HPLC analysis to profile lipid content. Values are mean ± S.E.M. Statistical analysis was performed using one-way ANOVA (Turkey’s post-tests). * p

    Journal: Oncotarget

    Article Title: PPARγ sumoylation-mediated lipid accumulation in lung cancer

    doi: 10.18632/oncotarget.19700

    Figure Lengend Snippet: PPARγ activation induces de novo lipid synthesis in a sumoylation dependent manner (A) Sumoylation of endogenous PPARγ in Calu6. PPARγ immunoprecipitates from cell lysates treated with 30 μM of pioglitazone for 24 hours were subjected to western blot using indicated antibodies. (B, C) Sumoylation of PPARγWT or PPARγSUMO (K107, 395R). (B) HEK293 cells were co-transfected with plasmids expressing BirA, Flag-SUMO1, and BLRP-PPARγ WT or BLRP-PPARγ SUMO, and further treated with 3 μM of pioglitazone for 24 hours. Streptavidine (strep) immunoprecipitates were subjected to western blot using indicated antibodies. (C) Sumoylation of PPARγ in HBEC cells with stable expression of BLRP-PPARγ WT or BLRP-PPARγ SUMO upon tetracycline induction. Streptavidine immunoprecipitates from HBEC cells infected with pAd-BirA were subjected to western blot using indicated antibodies. (D) ORO lipid staining in HBEC cells. HBEC cells under tetracycline (Tet) ON or OFF conditions were treated with pioglitazone (3 μM) for 5 days in media containing 5% CS, followed by ORO staining to detect lipid droplet accumulation. (E) Metabolic profiling of fatty acids upon ligand activation of PPARγ. HBEC cells were treated with pioglitazone (3 μM) for 5 days, followed by free fatty acid extraction and HPLC analysis to profile lipid content. Values are mean ± S.E.M. Statistical analysis was performed using one-way ANOVA (Turkey’s post-tests). * p

    Article Snippet: Sumoylation assay HEK293 cells transfected with BLRP-tagged PPARγWT or PPARγSUMO (K107, 395R), biotin-protein ligase (BirA) and Flag-tagged SUMO1, or HBEC cells infected with BirA adenovirus were lysed in immunoprecipitation (IP) lysis buffer containing 10 mM tris-HCl pH 7.5, 150 mM NaCl, 10 mM phosphate buffer, 1% triton X-100, 20 mM N-ethylmaleimide (Sigma) and protease inhibitor cocktail (Roche).

    Techniques: Activation Assay, Western Blot, Transfection, Expressing, Infection, Staining, High Performance Liquid Chromatography

    DHHC21 mediates TCR-induced S-acylation of signaling proteins. (A) Overview of the acyl-resin assisted capture (Acyl-RAC) assay. Free cysteine thiols (-SH) are irreversibly blocked by S-methyl methanethiosulfonate (MMTS) following cell lysis. Thioester bonds between cysteines and acyl groups are then specifically cleaved by neutral hydroxylamine (HA). The newly formed free thiol groups are captured by the thiol-reactive sepharose and S-acylated proteins detected by immunoblotting. (B) TCR-induced protein S-acylation. CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with cross-linked anti-CD3/CD28 antibodies for indicated times and protein S-acylation (acyl-) was determined using Acyl-RAC assay. Samples not treated with hydroxylamine (-HA) were used as a negative control. β-Actin, a known S-acylated protein ( 32 ), was used as a loading control.

    Journal: bioRxiv

    Article Title: Calcium-dependent protein acyltransferase DHHC21 controls activation of CD4+ T cells

    doi: 10.1101/2020.09.01.277947

    Figure Lengend Snippet: DHHC21 mediates TCR-induced S-acylation of signaling proteins. (A) Overview of the acyl-resin assisted capture (Acyl-RAC) assay. Free cysteine thiols (-SH) are irreversibly blocked by S-methyl methanethiosulfonate (MMTS) following cell lysis. Thioester bonds between cysteines and acyl groups are then specifically cleaved by neutral hydroxylamine (HA). The newly formed free thiol groups are captured by the thiol-reactive sepharose and S-acylated proteins detected by immunoblotting. (B) TCR-induced protein S-acylation. CD4 + T cells from WT or Zdhhc21 dep mice were stimulated with cross-linked anti-CD3/CD28 antibodies for indicated times and protein S-acylation (acyl-) was determined using Acyl-RAC assay. Samples not treated with hydroxylamine (-HA) were used as a negative control. β-Actin, a known S-acylated protein ( 32 ), was used as a loading control.

    Article Snippet: The following reagents were purchased from Sigma-Aldrich: Protein A Sepharose (Cat. p6649), Hydroxylamine (Cat. 55459), S-methyl methanethiosulfonate (MMTS) (Cat. 208795), n-Dodecyl β-D-maltoside (DDM) (Cat. D4641), Thiopropyl-Sepharose 6B (Cat. T8387), Poly-L-lysine (Cat. P8920), Phosphatase Inhibitor Cocktail 2 (Cat. P5726), Complete Protease Inhibitor Cocktail tablets (Cat. 11836170001).

    Techniques: Lysis, Mouse Assay, Negative Control

    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