pierce ecl western blotting substrate Thermo Fisher Search Results


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    Thermo Fisher ecl western blotting substrate
    Pmp18.1 activates MyD88, NFκB and Caspase-1 expression in BMDCs. Cell lysates were prepared from BMDCs incubated with Pmp18.1 (10 μg/ml) for 1, 2, or 24 h. Protein extracts (40 μg/well) were separated by SDS-PAGE and detected by immunoblotting analysis incorporating the Pierce™ <t>ECL</t> Western Blotting Substrate using antibodies to the corresponding proteins. The proteins bands were visualized using the <t>ImageQuant</t> LAS-4000 imaging system. Protein levels, normalized to GAPDH, were then quantified using Image J software. The data shows the expression levels and fold induction of TLR4 (A) , MyD88 (B) , NF-κB p50 (C) , and Caspase-1 (D) . Each experiment was repeated at least two times with similar findings. For NF-κB p65 nuclear migration, BMDCs were incubated for 24 h with rPmp18.1 and nuclear NF-κB p65 was detected by confocal microscopy using a FITC conjugated monoclonal antibody to NF-κB p65 (clone D14E12, Cell Signaling Technology). (E) NF-κB p65 unit was stained in green, and nuclei were visualized by DAPI counterstain (blue). Note the diffuse distribution of NF-κB p65 (green) in the nucleus. The statistical significance difference between two groups was evaluated by Student's t -test and between more than two groups by the one-way analysis of variance (ANOVA). Differences were considered to be statistically significant at * p
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    Thermo Fisher pierce ecl western blotting substrate
    5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse <t>IgG</t> conjugated with horseradish peroxidase (HRP) and Pierce <t>ECL</t> Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P
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    Thermo Fisher ecl
    Hepatic LXR protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-LXR Ab (1:1000), incubation with secondary Ab conjugated with <t>HRP</t> and bands visualization after <t>ECL</t> reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error
    Ecl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 10427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher chemiluminescence western blot substrate
    Hepatic LXR protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-LXR Ab (1:1000), incubation with secondary Ab conjugated with <t>HRP</t> and bands visualization after <t>ECL</t> reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error
    Chemiluminescence Western Blot Substrate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher chemiluminescence reagent
    Hepatic LXR protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-LXR Ab (1:1000), incubation with secondary Ab conjugated with <t>HRP</t> and bands visualization after <t>ECL</t> reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error
    Chemiluminescence Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher supersignal west pico chemiluminescent substrate
    (A) Overexpression of constitutively active Akt protects LNCaP cells from HMBME-induced cell growth inhibition. Subconfluent LNCaP cells were transfected with either pCMVMyrAkt (an activated form of Akt with the Src myristoylation signal fused in-frame to the c-Akt coding sequence) or control vector (pCMV) using Lipofectamin (Invitrogen) in triplicate dishes. Forty-eight hours following transfection, cells were treated either with 25 µM HMBME or solvent control. Both floating and adherent cells were collected after 2 hours of treatment and assessed for cell viability by trypan blue exclusion assay. The data shown here are average ± SD of three independent experiments. (B) Overexpression of constitutively active Akt protects LNCaP cells from undergoing apoptosis following treatment with HMBME. Experiments were conducted essentially as described in (A). Both floating and adherent cells were collected after 2 hours of treatment and assessed for apoptosis as described in Materials and Methods section. The data shown here are average ± SD of two independent transfections. (C) Influence of HMBME on transcriptional activity of the NFκB promoter. Transient transfections were performed with pNFκB reporter plasmid (1 µg/well) and pRL-TK plasmid (50 ng/well; renilla luciferase for normalization) as described in Materials and Methods section using Lipofectin reagent. Forty-eight hours after transfection, cells were treated with HMBME (25 µM) for 2 hours. Firefly and renilla luciferase activity was measured in the extracts prepared from these using Dual Luciferase Reporter Assay System (Promega) in duplicate samples containing equal amounts of protein. Renilla luciferase activity was used to normalize for transfection efficiency. Results are expressed as the ratio of firefly luciferase/renilla luciferase at equal amounts of protein. The data shown here are a representative experiment that was performed for four times with two different preparations of plasmid. For cotransfection experiments, indicated expression plasmids (1 µg/well) were included along with the pNFκB reporter plasmid. The data shown here are a representative experiment that was performed for four times with two different preparations of plasmid. (D) EMSA of nuclear extracts prepared from control and HMBME-treated cells. Nuclear extract (5 µg) was incubated with approximately 0.2 ng of NFκB consensus oligonucleotide as radiolabeled probe as described in Materials and Methods section and the DNA-protein complexes were resolved on a 4% nondenaturing gel by electrophoresis and subject to autoradiography. (E) Identification of protein components of NFκB DNA-binding activity. Nuclear extracts prepared from LNCaP cells were preincubated with p65, p50, or NRS for 30 minutes on ice. These extracts were used in EMSA with NFkB probe as described in legends for (C). Supershifted complex is indicated as p65/p50 complex and NS indicates nonspecific band. (F) Western blot analysis of whole cell extracts from LNCaP cells following treatment with HMBME. Twenty-five micrograms of extract from control or HMBME-treated cells was fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated for 2 or 3 hours with the antibody p65. This was followed by incubation with secondary horseradish peroxidase-conjugated antirabbit IgG antibody (Sigma) in blocking solution. Bound antibody was detected by <t>Supersignal</t> West Pico Chemiluminescent Substrate, following the manufacturer's directions (Pierce). The blot shown here is a representative blot of three independent experiments.
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    Thermo Fisher ecl kit
    (A) Overexpression of constitutively active Akt protects LNCaP cells from HMBME-induced cell growth inhibition. Subconfluent LNCaP cells were transfected with either pCMVMyrAkt (an activated form of Akt with the Src myristoylation signal fused in-frame to the c-Akt coding sequence) or control vector (pCMV) using Lipofectamin (Invitrogen) in triplicate dishes. Forty-eight hours following transfection, cells were treated either with 25 µM HMBME or solvent control. Both floating and adherent cells were collected after 2 hours of treatment and assessed for cell viability by trypan blue exclusion assay. The data shown here are average ± SD of three independent experiments. (B) Overexpression of constitutively active Akt protects LNCaP cells from undergoing apoptosis following treatment with HMBME. Experiments were conducted essentially as described in (A). Both floating and adherent cells were collected after 2 hours of treatment and assessed for apoptosis as described in Materials and Methods section. The data shown here are average ± SD of two independent transfections. (C) Influence of HMBME on transcriptional activity of the NFκB promoter. Transient transfections were performed with pNFκB reporter plasmid (1 µg/well) and pRL-TK plasmid (50 ng/well; renilla luciferase for normalization) as described in Materials and Methods section using Lipofectin reagent. Forty-eight hours after transfection, cells were treated with HMBME (25 µM) for 2 hours. Firefly and renilla luciferase activity was measured in the extracts prepared from these using Dual Luciferase Reporter Assay System (Promega) in duplicate samples containing equal amounts of protein. Renilla luciferase activity was used to normalize for transfection efficiency. Results are expressed as the ratio of firefly luciferase/renilla luciferase at equal amounts of protein. The data shown here are a representative experiment that was performed for four times with two different preparations of plasmid. For cotransfection experiments, indicated expression plasmids (1 µg/well) were included along with the pNFκB reporter plasmid. The data shown here are a representative experiment that was performed for four times with two different preparations of plasmid. (D) EMSA of nuclear extracts prepared from control and HMBME-treated cells. Nuclear extract (5 µg) was incubated with approximately 0.2 ng of NFκB consensus oligonucleotide as radiolabeled probe as described in Materials and Methods section and the DNA-protein complexes were resolved on a 4% nondenaturing gel by electrophoresis and subject to autoradiography. (E) Identification of protein components of NFκB DNA-binding activity. Nuclear extracts prepared from LNCaP cells were preincubated with p65, p50, or NRS for 30 minutes on ice. These extracts were used in EMSA with NFkB probe as described in legends for (C). Supershifted complex is indicated as p65/p50 complex and NS indicates nonspecific band. (F) Western blot analysis of whole cell extracts from LNCaP cells following treatment with HMBME. Twenty-five micrograms of extract from control or HMBME-treated cells was fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated for 2 or 3 hours with the antibody p65. This was followed by incubation with secondary horseradish peroxidase-conjugated antirabbit IgG antibody (Sigma) in blocking solution. Bound antibody was detected by <t>Supersignal</t> West Pico Chemiluminescent Substrate, following the manufacturer's directions (Pierce). The blot shown here is a representative blot of three independent experiments.
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    Image Search Results


    Pmp18.1 activates MyD88, NFκB and Caspase-1 expression in BMDCs. Cell lysates were prepared from BMDCs incubated with Pmp18.1 (10 μg/ml) for 1, 2, or 24 h. Protein extracts (40 μg/well) were separated by SDS-PAGE and detected by immunoblotting analysis incorporating the Pierce™ ECL Western Blotting Substrate using antibodies to the corresponding proteins. The proteins bands were visualized using the ImageQuant LAS-4000 imaging system. Protein levels, normalized to GAPDH, were then quantified using Image J software. The data shows the expression levels and fold induction of TLR4 (A) , MyD88 (B) , NF-κB p50 (C) , and Caspase-1 (D) . Each experiment was repeated at least two times with similar findings. For NF-κB p65 nuclear migration, BMDCs were incubated for 24 h with rPmp18.1 and nuclear NF-κB p65 was detected by confocal microscopy using a FITC conjugated monoclonal antibody to NF-κB p65 (clone D14E12, Cell Signaling Technology). (E) NF-κB p65 unit was stained in green, and nuclei were visualized by DAPI counterstain (blue). Note the diffuse distribution of NF-κB p65 (green) in the nucleus. The statistical significance difference between two groups was evaluated by Student's t -test and between more than two groups by the one-way analysis of variance (ANOVA). Differences were considered to be statistically significant at * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways

    doi: 10.3389/fcimb.2017.00514

    Figure Lengend Snippet: Pmp18.1 activates MyD88, NFκB and Caspase-1 expression in BMDCs. Cell lysates were prepared from BMDCs incubated with Pmp18.1 (10 μg/ml) for 1, 2, or 24 h. Protein extracts (40 μg/well) were separated by SDS-PAGE and detected by immunoblotting analysis incorporating the Pierce™ ECL Western Blotting Substrate using antibodies to the corresponding proteins. The proteins bands were visualized using the ImageQuant LAS-4000 imaging system. Protein levels, normalized to GAPDH, were then quantified using Image J software. The data shows the expression levels and fold induction of TLR4 (A) , MyD88 (B) , NF-κB p50 (C) , and Caspase-1 (D) . Each experiment was repeated at least two times with similar findings. For NF-κB p65 nuclear migration, BMDCs were incubated for 24 h with rPmp18.1 and nuclear NF-κB p65 was detected by confocal microscopy using a FITC conjugated monoclonal antibody to NF-κB p65 (clone D14E12, Cell Signaling Technology). (E) NF-κB p65 unit was stained in green, and nuclei were visualized by DAPI counterstain (blue). Note the diffuse distribution of NF-κB p65 (green) in the nucleus. The statistical significance difference between two groups was evaluated by Student's t -test and between more than two groups by the one-way analysis of variance (ANOVA). Differences were considered to be statistically significant at * p

    Article Snippet: After washing five times with PBST, the membrane was incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (Southern Biotech, Birmingham, AL) at room temperature for 1 h. Proteins were detected by addition of the Pierce™ ECL Western Blotting Substrate and visualized using the ImageQuant LAS-4000 (Thermo Fisher Scientific, Rockford, IL).

    Techniques: Expressing, Incubation, SDS Page, Western Blot, Imaging, Software, Migration, Confocal Microscopy, Staining

    5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P

    Journal: Scientific Reports

    Article Title: Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors

    doi: 10.1038/srep24942

    Figure Lengend Snippet: 5f restored neural tube development and marker expression in chick embryos on EDD 5. Glucose concentration ( A ) and Pax3 protein expression ( B ) were determined in chick embryos. The plasma glucose concentration was measured using a glucose oxidase-coupled spectrophotometric assay kit. Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP. The abbreviations “CON”, “GLU”, “CL”, “CM”, “CH”, “EPA”, “EDA” mean “controlled”, “glucose treated”, “low concentration of 5f treated”, “mild concentration of 5f treated”, “high concentration of 5f treated”, “epalrestat treated”, “edaravone treated” groups, respectively. Values were expressed as mean ± SD in each group (n = 10). * P

    Article Snippet: Proteins were detected using the monoclonal antibody anti-Pax3 diluted 1:1000 (DSHB, USA) and visualized using anti-mouse IgG conjugated with horseradish peroxidase (HRP) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) as the substrate of HRP.

    Techniques: Marker, Expressing, Concentration Assay, Spectrophotometric Assay, Western Blot

    Hepatic LXR protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-LXR Ab (1:1000), incubation with secondary Ab conjugated with HRP and bands visualization after ECL reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error

    Journal: Lipids in Health and Disease

    Article Title: Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta

    doi: 10.1186/s12944-015-0093-3

    Figure Lengend Snippet: Hepatic LXR protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-LXR Ab (1:1000), incubation with secondary Ab conjugated with HRP and bands visualization after ECL reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error

    Article Snippet: Membranes were incubated with HRP-conjugated antibody and reacted against ECL (Super Signal West Pico Chemiluminescent substract, Pierce, Rockford, IL, EUA).

    Techniques: Mouse Assay, Incubation

    Hepatic LDL receptor protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-LDL receptor Ab (1:1000), incubation with secondary Ab conjugated with HRP and bands visualization after ECL reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error

    Journal: Lipids in Health and Disease

    Article Title: Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta

    doi: 10.1186/s12944-015-0093-3

    Figure Lengend Snippet: Hepatic LDL receptor protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-LDL receptor Ab (1:1000), incubation with secondary Ab conjugated with HRP and bands visualization after ECL reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error

    Article Snippet: Membranes were incubated with HRP-conjugated antibody and reacted against ECL (Super Signal West Pico Chemiluminescent substract, Pierce, Rockford, IL, EUA).

    Techniques: Mouse Assay, Incubation

    Hepatic SR-BI protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-SR-BI Ab (1:1000), incubation with secondary Ab conjugated with HRP and bands visualization after ECL reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error

    Journal: Lipids in Health and Disease

    Article Title: Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta

    doi: 10.1186/s12944-015-0093-3

    Figure Lengend Snippet: Hepatic SR-BI protein level in trained and sedentary C57BL/6N wild type mice. Equal amounts of liver lysates were applied into a 10 % polyacrylamide gel. Immunoblotting was performed by using an anti-SR-BI Ab (1:1000), incubation with secondary Ab conjugated with HRP and bands visualization after ECL reaction. Each lane represents one animal sample. Data are expressed as mean values ± standard error

    Article Snippet: Membranes were incubated with HRP-conjugated antibody and reacted against ECL (Super Signal West Pico Chemiluminescent substract, Pierce, Rockford, IL, EUA).

    Techniques: Mouse Assay, Incubation

    (A) Overexpression of constitutively active Akt protects LNCaP cells from HMBME-induced cell growth inhibition. Subconfluent LNCaP cells were transfected with either pCMVMyrAkt (an activated form of Akt with the Src myristoylation signal fused in-frame to the c-Akt coding sequence) or control vector (pCMV) using Lipofectamin (Invitrogen) in triplicate dishes. Forty-eight hours following transfection, cells were treated either with 25 µM HMBME or solvent control. Both floating and adherent cells were collected after 2 hours of treatment and assessed for cell viability by trypan blue exclusion assay. The data shown here are average ± SD of three independent experiments. (B) Overexpression of constitutively active Akt protects LNCaP cells from undergoing apoptosis following treatment with HMBME. Experiments were conducted essentially as described in (A). Both floating and adherent cells were collected after 2 hours of treatment and assessed for apoptosis as described in Materials and Methods section. The data shown here are average ± SD of two independent transfections. (C) Influence of HMBME on transcriptional activity of the NFκB promoter. Transient transfections were performed with pNFκB reporter plasmid (1 µg/well) and pRL-TK plasmid (50 ng/well; renilla luciferase for normalization) as described in Materials and Methods section using Lipofectin reagent. Forty-eight hours after transfection, cells were treated with HMBME (25 µM) for 2 hours. Firefly and renilla luciferase activity was measured in the extracts prepared from these using Dual Luciferase Reporter Assay System (Promega) in duplicate samples containing equal amounts of protein. Renilla luciferase activity was used to normalize for transfection efficiency. Results are expressed as the ratio of firefly luciferase/renilla luciferase at equal amounts of protein. The data shown here are a representative experiment that was performed for four times with two different preparations of plasmid. For cotransfection experiments, indicated expression plasmids (1 µg/well) were included along with the pNFκB reporter plasmid. The data shown here are a representative experiment that was performed for four times with two different preparations of plasmid. (D) EMSA of nuclear extracts prepared from control and HMBME-treated cells. Nuclear extract (5 µg) was incubated with approximately 0.2 ng of NFκB consensus oligonucleotide as radiolabeled probe as described in Materials and Methods section and the DNA-protein complexes were resolved on a 4% nondenaturing gel by electrophoresis and subject to autoradiography. (E) Identification of protein components of NFκB DNA-binding activity. Nuclear extracts prepared from LNCaP cells were preincubated with p65, p50, or NRS for 30 minutes on ice. These extracts were used in EMSA with NFkB probe as described in legends for (C). Supershifted complex is indicated as p65/p50 complex and NS indicates nonspecific band. (F) Western blot analysis of whole cell extracts from LNCaP cells following treatment with HMBME. Twenty-five micrograms of extract from control or HMBME-treated cells was fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated for 2 or 3 hours with the antibody p65. This was followed by incubation with secondary horseradish peroxidase-conjugated antirabbit IgG antibody (Sigma) in blocking solution. Bound antibody was detected by Supersignal West Pico Chemiluminescent Substrate, following the manufacturer's directions (Pierce). The blot shown here is a representative blot of three independent experiments.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: 4-Hydroxy-3-Methoxybenzoic Acid Methyl Ester: A Curcumin Derivative Targets Akt/NF?B Cell Survival Signaling Pathway: Potential for Prostate Cancer Management

    doi:

    Figure Lengend Snippet: (A) Overexpression of constitutively active Akt protects LNCaP cells from HMBME-induced cell growth inhibition. Subconfluent LNCaP cells were transfected with either pCMVMyrAkt (an activated form of Akt with the Src myristoylation signal fused in-frame to the c-Akt coding sequence) or control vector (pCMV) using Lipofectamin (Invitrogen) in triplicate dishes. Forty-eight hours following transfection, cells were treated either with 25 µM HMBME or solvent control. Both floating and adherent cells were collected after 2 hours of treatment and assessed for cell viability by trypan blue exclusion assay. The data shown here are average ± SD of three independent experiments. (B) Overexpression of constitutively active Akt protects LNCaP cells from undergoing apoptosis following treatment with HMBME. Experiments were conducted essentially as described in (A). Both floating and adherent cells were collected after 2 hours of treatment and assessed for apoptosis as described in Materials and Methods section. The data shown here are average ± SD of two independent transfections. (C) Influence of HMBME on transcriptional activity of the NFκB promoter. Transient transfections were performed with pNFκB reporter plasmid (1 µg/well) and pRL-TK plasmid (50 ng/well; renilla luciferase for normalization) as described in Materials and Methods section using Lipofectin reagent. Forty-eight hours after transfection, cells were treated with HMBME (25 µM) for 2 hours. Firefly and renilla luciferase activity was measured in the extracts prepared from these using Dual Luciferase Reporter Assay System (Promega) in duplicate samples containing equal amounts of protein. Renilla luciferase activity was used to normalize for transfection efficiency. Results are expressed as the ratio of firefly luciferase/renilla luciferase at equal amounts of protein. The data shown here are a representative experiment that was performed for four times with two different preparations of plasmid. For cotransfection experiments, indicated expression plasmids (1 µg/well) were included along with the pNFκB reporter plasmid. The data shown here are a representative experiment that was performed for four times with two different preparations of plasmid. (D) EMSA of nuclear extracts prepared from control and HMBME-treated cells. Nuclear extract (5 µg) was incubated with approximately 0.2 ng of NFκB consensus oligonucleotide as radiolabeled probe as described in Materials and Methods section and the DNA-protein complexes were resolved on a 4% nondenaturing gel by electrophoresis and subject to autoradiography. (E) Identification of protein components of NFκB DNA-binding activity. Nuclear extracts prepared from LNCaP cells were preincubated with p65, p50, or NRS for 30 minutes on ice. These extracts were used in EMSA with NFkB probe as described in legends for (C). Supershifted complex is indicated as p65/p50 complex and NS indicates nonspecific band. (F) Western blot analysis of whole cell extracts from LNCaP cells following treatment with HMBME. Twenty-five micrograms of extract from control or HMBME-treated cells was fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated for 2 or 3 hours with the antibody p65. This was followed by incubation with secondary horseradish peroxidase-conjugated antirabbit IgG antibody (Sigma) in blocking solution. Bound antibody was detected by Supersignal West Pico Chemiluminescent Substrate, following the manufacturer's directions (Pierce). The blot shown here is a representative blot of three independent experiments.

    Article Snippet: Bound antibody was detected by enhanced chemiluminescence using Supersignal West Pico Chemiluminescent Substrate, following the manufacturer's directions (Pierce, Rockford, IL).

    Techniques: Over Expression, Inhibition, Transfection, Sequencing, Plasmid Preparation, Trypan Blue Exclusion Assay, Activity Assay, Luciferase, Reporter Assay, Cotransfection, Expressing, Incubation, Nucleic Acid Electrophoresis, Autoradiography, Binding Assay, Western Blot, SDS Page, Blocking Assay