Structured Review

Santa Cruz Biotechnology protease inhibitors
Protease Inhibitors, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/protease inhibitors/product/Santa Cruz Biotechnology
Average 92 stars, based on 44 article reviews
Price from $9.99 to $1999.99
protease inhibitors - by Bioz Stars, 2020-09
92/100 stars

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Related Articles

Transfection:

Article Title: A novel transcript of the LMO2 gene, LMO2-c, is regulated by GATA-1 and PU.1 and encodes an antagonist of LMO2
Article Snippet: .. After transfection, cells were washed twice with phosphate-buffered saline, lysed for 30 min in lysis buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 1% Nonidet P-40) containing protease inhibitors (10 μ g/ml leupeptin, 10 μ g/ml pepstatin A and 10 μ g/ml aprotinin) and phosphatase inhibitors (1mM NaF and 1mM Na3 VO4 ) and clarified by centrifugation at 4°C for 30 min. Supernatants were precleared with protein G plus agarose (Santa Cruz Biotechnology) for 2 h at 4°C. ..

Radio Immunoprecipitation:

Article Title: Inhibitory Effects of Dopamine Receptor D1 Agonist on Mammary Tumor and Bone Metastasis
Article Snippet: .. Western blot analysis Cells were lysed in a radio-immunoprecipitation assay (RIPA) buffer with protease inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phosphatase inhibitors (Calbiochem, Billerica, MA, USA). .. Isolated proteins were fractionated using 10–15% SDS gels and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA).

Magnetic Beads:

Article Title: Activation of the Smk1 Mitogen-Activated Protein Kinase by Developmentally Regulated Autophosphorylation
Article Snippet: .. Smk1-HA and Smk1-T207A-HA were transcribed and translated in a 100-μl TNT reaction volume (Promega) as per the manufacturer's protocol, diluted 1:10 in phosphate-buffered saline supplemented with 0.05% Tween 20 and protease inhibitors at the concentration specified by Schindler and Winter , and immunoprecipitated using rabbit anti-HA antisera (Santa Cruz Biotechnology Inc.) and protein A/G magnetic beads (Pierce). ..

Centrifugation:

Article Title: A novel transcript of the LMO2 gene, LMO2-c, is regulated by GATA-1 and PU.1 and encodes an antagonist of LMO2
Article Snippet: .. After transfection, cells were washed twice with phosphate-buffered saline, lysed for 30 min in lysis buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 1% Nonidet P-40) containing protease inhibitors (10 μ g/ml leupeptin, 10 μ g/ml pepstatin A and 10 μ g/ml aprotinin) and phosphatase inhibitors (1mM NaF and 1mM Na3 VO4 ) and clarified by centrifugation at 4°C for 30 min. Supernatants were precleared with protein G plus agarose (Santa Cruz Biotechnology) for 2 h at 4°C. ..

Incubation:

Article Title: The Transcription Factor COL12 Is a Substrate of the COP1/SPA E3 Ligase and Regulates Flowering Time and Plant Architecture 1
Article Snippet: .. TnT-produced bait and prey proteins were added to 0.1 mL of binding buffer (20 m m Tris-HCl pH 7.5, 150 m m NaCl, 1 m m dithiothreitol, 0.1% Tween 20) with protease inhibitors (C o mplete, EDTA-free) and incubated on a rotating platform at 4°C for 2 to 3 h Following addition of 1 μg of monoclonal antibody against GAD (Santa Cruz Biotechnology) incubation continued for 1 more hour. .. Subsequently, 20 μL of protein A-coated magnetic beads (Dynal) was added.

Immunoprecipitation:

Article Title: Molecular and Structural Insight into proNGF Engagement of p75NTR and Sortilin
Article Snippet: .. Cells were then harvested in Ca2+ -supplemented (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40 and 2 mM CaCl2 ) or Ca2+ -depleted lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40 and 2 mM EDTA) with protease inhibitors, and cell lysates (0.3 mg) were immunoprecipitated with anti-Myc affinity matrix (Santa Cruz Biotechnology). .. To detect sortilin-proNGF complexes in the cellular media, culture media (Opti-MEM I; 1.5 ml) was harvested, cleared by centrifugation, and immunoprecipitated with a biotinylated sortilin antibody (BAF2934, R & D Systems) followed by streptavidin pull-down in Ca2+ -supplemented (with 2 mM CaCl2 and protease inhibitors) or Ca2+ -depleted conditions (with 2 mM EDTA and protease inhibitors).

Article Title: Activation of the Smk1 Mitogen-Activated Protein Kinase by Developmentally Regulated Autophosphorylation
Article Snippet: .. Smk1-HA and Smk1-T207A-HA were transcribed and translated in a 100-μl TNT reaction volume (Promega) as per the manufacturer's protocol, diluted 1:10 in phosphate-buffered saline supplemented with 0.05% Tween 20 and protease inhibitors at the concentration specified by Schindler and Winter , and immunoprecipitated using rabbit anti-HA antisera (Santa Cruz Biotechnology Inc.) and protein A/G magnetic beads (Pierce). ..

Concentration Assay:

Article Title: Activation of the Smk1 Mitogen-Activated Protein Kinase by Developmentally Regulated Autophosphorylation
Article Snippet: .. Smk1-HA and Smk1-T207A-HA were transcribed and translated in a 100-μl TNT reaction volume (Promega) as per the manufacturer's protocol, diluted 1:10 in phosphate-buffered saline supplemented with 0.05% Tween 20 and protease inhibitors at the concentration specified by Schindler and Winter , and immunoprecipitated using rabbit anti-HA antisera (Santa Cruz Biotechnology Inc.) and protein A/G magnetic beads (Pierce). ..

Article Title: Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent
Article Snippet: .. Western blotting Total proteins were extracted from cells using protein extraction reagents containing protease inhibitors (Santa Cruz), and the concentration of protein extract was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). .. Equivalent amounts of protein from each sample were resolved by SDS-PAGE and subsequently transferred onto a PVDF membrane (Millipore).

Protein Extraction:

Article Title: Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent
Article Snippet: .. Western blotting Total proteins were extracted from cells using protein extraction reagents containing protease inhibitors (Santa Cruz), and the concentration of protein extract was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). .. Equivalent amounts of protein from each sample were resolved by SDS-PAGE and subsequently transferred onto a PVDF membrane (Millipore).

Western Blot:

Article Title: Inhibitory Effects of Dopamine Receptor D1 Agonist on Mammary Tumor and Bone Metastasis
Article Snippet: .. Western blot analysis Cells were lysed in a radio-immunoprecipitation assay (RIPA) buffer with protease inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phosphatase inhibitors (Calbiochem, Billerica, MA, USA). .. Isolated proteins were fractionated using 10–15% SDS gels and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA).

Article Title: Mouse bone marrow mesenchymal stem cells with distinct p53 statuses display differential characteristics
Article Snippet: .. Western blotting Total protein was extracted from p53+/+ , p53+/− and p53−/− mBM-MSCs using RIPA buffer supplemented with protease inhibitors (Santa Cruz Biotechnology, Inc.). .. Total protein concentration was quantified using a BCA assay kit (Thermo Fisher Scientific, Inc.) and 40 µg protein/lane was separated by 10% SDS-PAGE.

Article Title: Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent
Article Snippet: .. Western blotting Total proteins were extracted from cells using protein extraction reagents containing protease inhibitors (Santa Cruz), and the concentration of protein extract was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). .. Equivalent amounts of protein from each sample were resolved by SDS-PAGE and subsequently transferred onto a PVDF membrane (Millipore).

Lysis:

Article Title: Molecular and Structural Insight into proNGF Engagement of p75NTR and Sortilin
Article Snippet: .. Cells were then harvested in Ca2+ -supplemented (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40 and 2 mM CaCl2 ) or Ca2+ -depleted lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40 and 2 mM EDTA) with protease inhibitors, and cell lysates (0.3 mg) were immunoprecipitated with anti-Myc affinity matrix (Santa Cruz Biotechnology). .. To detect sortilin-proNGF complexes in the cellular media, culture media (Opti-MEM I; 1.5 ml) was harvested, cleared by centrifugation, and immunoprecipitated with a biotinylated sortilin antibody (BAF2934, R & D Systems) followed by streptavidin pull-down in Ca2+ -supplemented (with 2 mM CaCl2 and protease inhibitors) or Ca2+ -depleted conditions (with 2 mM EDTA and protease inhibitors).

Article Title: A novel transcript of the LMO2 gene, LMO2-c, is regulated by GATA-1 and PU.1 and encodes an antagonist of LMO2
Article Snippet: .. After transfection, cells were washed twice with phosphate-buffered saline, lysed for 30 min in lysis buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 1% Nonidet P-40) containing protease inhibitors (10 μ g/ml leupeptin, 10 μ g/ml pepstatin A and 10 μ g/ml aprotinin) and phosphatase inhibitors (1mM NaF and 1mM Na3 VO4 ) and clarified by centrifugation at 4°C for 30 min. Supernatants were precleared with protein G plus agarose (Santa Cruz Biotechnology) for 2 h at 4°C. ..

Binding Assay:

Article Title: The Transcription Factor COL12 Is a Substrate of the COP1/SPA E3 Ligase and Regulates Flowering Time and Plant Architecture 1
Article Snippet: .. TnT-produced bait and prey proteins were added to 0.1 mL of binding buffer (20 m m Tris-HCl pH 7.5, 150 m m NaCl, 1 m m dithiothreitol, 0.1% Tween 20) with protease inhibitors (C o mplete, EDTA-free) and incubated on a rotating platform at 4°C for 2 to 3 h Following addition of 1 μg of monoclonal antibody against GAD (Santa Cruz Biotechnology) incubation continued for 1 more hour. .. Subsequently, 20 μL of protein A-coated magnetic beads (Dynal) was added.

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  • 90
    Santa Cruz Biotechnology goat anti nadph oxidase 2
    Tart cherry consumption reduced inflammation in the hippocampus. The results shown represent western blot ( top ) of inflammatory markers (glial fibrillary acidic protein [GFAP], <t>NADPH</t> <t>oxidase-2</t> [NOX-2], and cyclooxygenase-2 [COX-2]) in the hippocampus of control ( opened bar ) and 2 % tart cherry-supplemented group ( hatched bar ), and densitometry analysis ( bottom ) of the immunoreactive bands normalized to β-actin, with results represented as mean ± SEM; n = 7/group. Asterisk indicates statistically significant difference between the groups ( p
    Goat Anti Nadph Oxidase 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti nadph oxidase 2/product/Santa Cruz Biotechnology
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti nadph oxidase 2 - by Bioz Stars, 2020-09
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    89
    Santa Cruz Biotechnology anti hbx
    Reciprocal <t>immunoprecipitation</t> of HepG2X and HepG2CAT protein extracts ( A ) with anti-mSin3A and western blot detection of <t>HBx</t> and ( B ) with anti-HBx and western blot detection of mSin3A. Lane 1 in panel A is a nuclear extract of HepG2X cells (70 μg). Immunofluorescent staining of HepG2X cells with anti-HBx ( C ), mSin3A ( D ), and DAPI ( E ). ( F ) is a merged image of HBx and mSin3A staining showing co-localization in the nucleus (arrow).
    Anti Hbx, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hbx/product/Santa Cruz Biotechnology
    Average 89 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti hbx - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    88
    Santa Cruz Biotechnology anti tβri antibody
    Cooperativity between NS3 and TNF-α in the stimulation of TGF-β1, collagen α1(I), and <t>TβRI</t> expression. (A) Effect on TGF-β1 and collagen α1(I) mRNA expression in LX-2 cells. The cells were stimulated with 50 μg/ml of NS3 for 12 hours. Total cellular RNA was isolated and reverse transcribed to cDNA, and real-time PCR was performed as described in the Methods section. * p
    Anti Tβri Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti tβri antibody/product/Santa Cruz Biotechnology
    Average 88 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    anti tβri antibody - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    88
    Santa Cruz Biotechnology rabbit anti human tslp
    <t>TSLP</t> gene expression is up-regulated in <t>Sp1-silenced</t> NHK depending on enhanced protease activity a) TSLP mRNA is significantly increased at days three and four following Sp1 silencing as compared to NHK transfected with scrambled siRNA. b) TSLP protein was detected by western-blot in NHK four days after transfection of siRNA duplexes. c) NHK cells were transfected with scrambled siRNA and Sp1 siRNA duplexes in the absence and presence of protease inhibitor AEBSF for three days. mRNA levels of TSLP were measured by quantitative real-time PCR. d) NHK cells were transfected with scrambled siRNA and Sp1 siRNA duplexes in the absence and presence of six anti-KLK neutralizing antibodies and isotype control IgG for three days (see methods for details). mRNA levels of TSLP were measured by quantitative real-time PCR. Data are presented as mean ± s.e.m of triplicate experiments. ** P
    Rabbit Anti Human Tslp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti human tslp/product/Santa Cruz Biotechnology
    Average 88 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    rabbit anti human tslp - by Bioz Stars, 2020-09
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    Image Search Results


    Tart cherry consumption reduced inflammation in the hippocampus. The results shown represent western blot ( top ) of inflammatory markers (glial fibrillary acidic protein [GFAP], NADPH oxidase-2 [NOX-2], and cyclooxygenase-2 [COX-2]) in the hippocampus of control ( opened bar ) and 2 % tart cherry-supplemented group ( hatched bar ), and densitometry analysis ( bottom ) of the immunoreactive bands normalized to β-actin, with results represented as mean ± SEM; n = 7/group. Asterisk indicates statistically significant difference between the groups ( p

    Journal: Age

    Article Title: Tart cherry supplementation improves working memory, hippocampal inflammation, and autophagy in aged rats

    doi: 10.1007/s11357-016-9945-7

    Figure Lengend Snippet: Tart cherry consumption reduced inflammation in the hippocampus. The results shown represent western blot ( top ) of inflammatory markers (glial fibrillary acidic protein [GFAP], NADPH oxidase-2 [NOX-2], and cyclooxygenase-2 [COX-2]) in the hippocampus of control ( opened bar ) and 2 % tart cherry-supplemented group ( hatched bar ), and densitometry analysis ( bottom ) of the immunoreactive bands normalized to β-actin, with results represented as mean ± SEM; n = 7/group. Asterisk indicates statistically significant difference between the groups ( p

    Article Snippet: The following chemicals and antibodies were used in this study: complete Mini EDTA-free protease inhibitor (Roche Diagnostic, Indianapolis, IN); PMSF (Sigma-Aldrich, St. Louis, MO); 30 % acrylamide/bis solution, 29:1, TEMED, 4× Laemmli sample buffer, 10× Tris/glycine/SDS buffer, 10× Tris/glycine buffer, polyvinyl difluoride (PVDF) membrane (Bio-Rad, Hercules, CA); Rapid Block (Amresco, Solon, OH); goat-anti-NADPH oxidase-2 (NOX-2), goat-anti-cyclooxygenase-2 (COX-2), goat-anti-β-actin (Santa Cruz Biotechnology, Dallas, TX); goat-anti-glial fibrillary acidic protein (GFAP) (EMD Millipore, Billerica, MA); rabbit-anti-phosphorylated mammalian target of rapamycin (pmTOR), rabbit-anti-Beclin1, and mouse-anti-p62/SQSTM1 (Cell Signaling Technology, Danvers, MA); ECL Plus reagents (GE Healthcare Life Sciences, Piscataway, NJ).

    Techniques: Western Blot

    Reciprocal immunoprecipitation of HepG2X and HepG2CAT protein extracts ( A ) with anti-mSin3A and western blot detection of HBx and ( B ) with anti-HBx and western blot detection of mSin3A. Lane 1 in panel A is a nuclear extract of HepG2X cells (70 μg). Immunofluorescent staining of HepG2X cells with anti-HBx ( C ), mSin3A ( D ), and DAPI ( E ). ( F ) is a merged image of HBx and mSin3A staining showing co-localization in the nucleus (arrow).

    Journal: Oncogene

    Article Title: Epigenetic repression of E-cadherin expression by Hepatitis B virus x Antigen (HBx) in liver cancer

    doi: 10.1038/onc.2011.255

    Figure Lengend Snippet: Reciprocal immunoprecipitation of HepG2X and HepG2CAT protein extracts ( A ) with anti-mSin3A and western blot detection of HBx and ( B ) with anti-HBx and western blot detection of mSin3A. Lane 1 in panel A is a nuclear extract of HepG2X cells (70 μg). Immunofluorescent staining of HepG2X cells with anti-HBx ( C ), mSin3A ( D ), and DAPI ( E ). ( F ) is a merged image of HBx and mSin3A staining showing co-localization in the nucleus (arrow).

    Article Snippet: Immunoprecipitation (IP) Protein extracts (500 μg) were incubated with anti-HBx (Santa Cruz) or anti-mSin3a (Abcam) and protein G sepharose beads (GE Healthcare, Uppsala, Sweden), in HNTG buffer (20mM Hepes pH 7.5, 150mM NaCl, 0.1% Triton X-100, 10% glycerol, and protease inhibitor cocktail [Cell Signaling, Danvers, MA]) overnight at 4°C.

    Techniques: Immunoprecipitation, Western Blot, Staining

    Representative staining for ( A ) HBx, Snail-1, mSin3A, and E-cadherin in consecutive sections from a block of a patient with HBV infected liver (x400). Representative staining for mSin3A ( B ) and Snail-1 ( C ) from uninfected human liver (top photo in B and C ) and from a block of HCC (bottom photo in B and C ) (x400).

    Journal: Oncogene

    Article Title: Epigenetic repression of E-cadherin expression by Hepatitis B virus x Antigen (HBx) in liver cancer

    doi: 10.1038/onc.2011.255

    Figure Lengend Snippet: Representative staining for ( A ) HBx, Snail-1, mSin3A, and E-cadherin in consecutive sections from a block of a patient with HBV infected liver (x400). Representative staining for mSin3A ( B ) and Snail-1 ( C ) from uninfected human liver (top photo in B and C ) and from a block of HCC (bottom photo in B and C ) (x400).

    Article Snippet: Immunoprecipitation (IP) Protein extracts (500 μg) were incubated with anti-HBx (Santa Cruz) or anti-mSin3a (Abcam) and protein G sepharose beads (GE Healthcare, Uppsala, Sweden), in HNTG buffer (20mM Hepes pH 7.5, 150mM NaCl, 0.1% Triton X-100, 10% glycerol, and protease inhibitor cocktail [Cell Signaling, Danvers, MA]) overnight at 4°C.

    Techniques: Staining, Blocking Assay, Infection

    Cooperativity between NS3 and TNF-α in the stimulation of TGF-β1, collagen α1(I), and TβRI expression. (A) Effect on TGF-β1 and collagen α1(I) mRNA expression in LX-2 cells. The cells were stimulated with 50 μg/ml of NS3 for 12 hours. Total cellular RNA was isolated and reverse transcribed to cDNA, and real-time PCR was performed as described in the Methods section. * p

    Journal: Scientific Reports

    Article Title: HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-? type I receptor

    doi: 10.1038/srep03243

    Figure Lengend Snippet: Cooperativity between NS3 and TNF-α in the stimulation of TGF-β1, collagen α1(I), and TβRI expression. (A) Effect on TGF-β1 and collagen α1(I) mRNA expression in LX-2 cells. The cells were stimulated with 50 μg/ml of NS3 for 12 hours. Total cellular RNA was isolated and reverse transcribed to cDNA, and real-time PCR was performed as described in the Methods section. * p

    Article Snippet: Anti-TβRI antibody and anti-phospho-Smad3 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Immuno-Biological Laboratories (Gunma, Japan), respectively.

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

    HCV NS3 protease exerted TGF-β mimetic activity via the type I receptor. (A) TGF-β2 antigenicity of NS3. The indicated concentrations of recombinant NS3 protease were used in the TGF-β2 ELISA assays. (B) TGF-β mimetic activity of NS3 and its suppression by TβRI kinase inhibitors. (CAGA) 9 -Luc CCL64 cells were stimulated with 100 μg/ml of recombinant NS3 protease for 24 hours, with or without the indicated concentration of TβRI kinase inhibitor or the NS3 protease inhibitor VX-950 (telaprevir). After 24 hours, the cells were harvested and luciferase activity measured. † p

    Journal: Scientific Reports

    Article Title: HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-? type I receptor

    doi: 10.1038/srep03243

    Figure Lengend Snippet: HCV NS3 protease exerted TGF-β mimetic activity via the type I receptor. (A) TGF-β2 antigenicity of NS3. The indicated concentrations of recombinant NS3 protease were used in the TGF-β2 ELISA assays. (B) TGF-β mimetic activity of NS3 and its suppression by TβRI kinase inhibitors. (CAGA) 9 -Luc CCL64 cells were stimulated with 100 μg/ml of recombinant NS3 protease for 24 hours, with or without the indicated concentration of TβRI kinase inhibitor or the NS3 protease inhibitor VX-950 (telaprevir). After 24 hours, the cells were harvested and luciferase activity measured. † p

    Article Snippet: Anti-TβRI antibody and anti-phospho-Smad3 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Immuno-Biological Laboratories (Gunma, Japan), respectively.

    Techniques: Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay, Protease Inhibitor, Luciferase

    NS3 protease colocalized and directly interacted with TβRI on the surface of HCV-infected cells. (A) The detection of NS3 protease on the surface of HCV-infected Huh-7.5.1 cells. The cells were fixed, followed ± by permeabilization with Triton-X 100, and then stained with DAPI, anti-NS3 antibody, and anti-calnexin antibody. (B) The colocalization of NS3 protease with TβRI in HCV-infected Huh7.5.1 cells. The cells were fixed and stained with DAPI, anti-NS3 antibody, and anti-TβRI antibody, as described in the Methods section. Pearson's colocalization coefficient values were obtained from 4 randomly selected fields using the ZEN software. The results are shown as the mean ± SD and are representative of three independent experiments with similar results. (C) The detection of NS3-TβRI proximity by in situ PLA in HCV-infected Huh-7.5.1 cells. The red dots indicate interactions between NS3 protease and TβRI, and the nuclei were identified by DAPI staining. (D) The physical interaction of NS3protease with TβRI and TβRII. FLAG-tagged NS3protease was incubated with 6xHis-tagged TβRI and/or TβRII and immunoprecipitated. The coprecipitated proteins were visualized by immunoblotting using anti-His antibody. The gels were run under the same experimental conditions. Cropped blots are shown (full-length blots are presented in Supplementary Fig. S5 ). (E) The structural overview of the NS3protease. The indicated colored amino acids (site 1, red; site 2, magenta; and site 3, cyan) show the important residues within the putative binding sites to TβRI, and the sequences are presented in Table 1 . TGF-β mimetic activity of NS3 was inhibited in the presence of either anti-NS3 polyclonal antibodies against the predicted binding sites of TβRI (F), or anti-TβRI polyclonal antibodies against predicted binding sites of NS3 (G), and anti-NS3 monoclonal antibody against predicted binding site 3 of TβRI (H). Luciferase activities in (CAGA) 9 -Luc CCL64 cells were measured as before. Normal mouse IgG (Norm-IgG) was used as a negative control. The data are shown as the mean ± SD. † p

    Journal: Scientific Reports

    Article Title: HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-? type I receptor

    doi: 10.1038/srep03243

    Figure Lengend Snippet: NS3 protease colocalized and directly interacted with TβRI on the surface of HCV-infected cells. (A) The detection of NS3 protease on the surface of HCV-infected Huh-7.5.1 cells. The cells were fixed, followed ± by permeabilization with Triton-X 100, and then stained with DAPI, anti-NS3 antibody, and anti-calnexin antibody. (B) The colocalization of NS3 protease with TβRI in HCV-infected Huh7.5.1 cells. The cells were fixed and stained with DAPI, anti-NS3 antibody, and anti-TβRI antibody, as described in the Methods section. Pearson's colocalization coefficient values were obtained from 4 randomly selected fields using the ZEN software. The results are shown as the mean ± SD and are representative of three independent experiments with similar results. (C) The detection of NS3-TβRI proximity by in situ PLA in HCV-infected Huh-7.5.1 cells. The red dots indicate interactions between NS3 protease and TβRI, and the nuclei were identified by DAPI staining. (D) The physical interaction of NS3protease with TβRI and TβRII. FLAG-tagged NS3protease was incubated with 6xHis-tagged TβRI and/or TβRII and immunoprecipitated. The coprecipitated proteins were visualized by immunoblotting using anti-His antibody. The gels were run under the same experimental conditions. Cropped blots are shown (full-length blots are presented in Supplementary Fig. S5 ). (E) The structural overview of the NS3protease. The indicated colored amino acids (site 1, red; site 2, magenta; and site 3, cyan) show the important residues within the putative binding sites to TβRI, and the sequences are presented in Table 1 . TGF-β mimetic activity of NS3 was inhibited in the presence of either anti-NS3 polyclonal antibodies against the predicted binding sites of TβRI (F), or anti-TβRI polyclonal antibodies against predicted binding sites of NS3 (G), and anti-NS3 monoclonal antibody against predicted binding site 3 of TβRI (H). Luciferase activities in (CAGA) 9 -Luc CCL64 cells were measured as before. Normal mouse IgG (Norm-IgG) was used as a negative control. The data are shown as the mean ± SD. † p

    Article Snippet: Anti-TβRI antibody and anti-phospho-Smad3 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Immuno-Biological Laboratories (Gunma, Japan), respectively.

    Techniques: Infection, Staining, Software, In Situ, Proximity Ligation Assay, Incubation, Immunoprecipitation, Binding Assay, Activity Assay, Luciferase, Negative Control

    TSLP gene expression is up-regulated in Sp1-silenced NHK depending on enhanced protease activity a) TSLP mRNA is significantly increased at days three and four following Sp1 silencing as compared to NHK transfected with scrambled siRNA. b) TSLP protein was detected by western-blot in NHK four days after transfection of siRNA duplexes. c) NHK cells were transfected with scrambled siRNA and Sp1 siRNA duplexes in the absence and presence of protease inhibitor AEBSF for three days. mRNA levels of TSLP were measured by quantitative real-time PCR. d) NHK cells were transfected with scrambled siRNA and Sp1 siRNA duplexes in the absence and presence of six anti-KLK neutralizing antibodies and isotype control IgG for three days (see methods for details). mRNA levels of TSLP were measured by quantitative real-time PCR. Data are presented as mean ± s.e.m of triplicate experiments. ** P

    Journal: The Journal of investigative dermatology

    Article Title: Inhibition of Transcription Factor Specificity Protein 1 Alters the Gene Expression Profile of Keratinocytes Leading to Up-regulation of Kallikrein-related Peptidases and TSLP

    doi: 10.1038/jid.2011.202

    Figure Lengend Snippet: TSLP gene expression is up-regulated in Sp1-silenced NHK depending on enhanced protease activity a) TSLP mRNA is significantly increased at days three and four following Sp1 silencing as compared to NHK transfected with scrambled siRNA. b) TSLP protein was detected by western-blot in NHK four days after transfection of siRNA duplexes. c) NHK cells were transfected with scrambled siRNA and Sp1 siRNA duplexes in the absence and presence of protease inhibitor AEBSF for three days. mRNA levels of TSLP were measured by quantitative real-time PCR. d) NHK cells were transfected with scrambled siRNA and Sp1 siRNA duplexes in the absence and presence of six anti-KLK neutralizing antibodies and isotype control IgG for three days (see methods for details). mRNA levels of TSLP were measured by quantitative real-time PCR. Data are presented as mean ± s.e.m of triplicate experiments. ** P

    Article Snippet: Rabbit anti-human Sp1, Rabbit anti-human TSLP, and rabbit anti-human GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Expressing, Activity Assay, Transfection, Western Blot, Protease Inhibitor, Real-time Polymerase Chain Reaction