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Santa Cruz Biotechnology horseradish peroxidase linked anti mouse igg
Horseradish Peroxidase Linked Anti Mouse Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/horseradish peroxidase linked anti mouse igg/product/Santa Cruz Biotechnology
Average 80 stars, based on 3 article reviews
Price from $9.99 to $1999.99
horseradish peroxidase linked anti mouse igg - by Bioz Stars, 2020-02
80/100 stars

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Centrifugation:

Article Title: Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis
Article Snippet: The supernatant of spinal cord homogenates were obtained following a high speed centrifugation and processed in one of the two following ways, as described ( ): (i) The homogenates (10μg of total protein) were boiled in 2×SDS loading buffer that did not contain β-mercaptoethanol and then loaded on to 15% SDS polyacrylamide gels for western blot analysis. .. After washing, the rabbit antibody stained blots were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3000, Cell Signaling), while the mouse antibody stained blots were incubated with HRP-linked anti-mouse IgG (1:6000, Santa Cruz) followed by washing and then detection with an ECL-Plus detection kit (Amersham, NJ).

Article Title: Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-?B and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines
Article Snippet: After incubation overnight at −70°C, the lysates were sonicated (3 s on and 1 s off; total time, 6 min) and then subjected to centrifugation (20,000 × g ) at 4°C for 15 min to obtain a cytosolic fraction. .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse immunoglobulin G (Santa Cruz Biotechnology) or rabbit polyclonal antibody against I-κBα (1:1,000 dilution) (Santa Cruz Biotechnology) for 1 h. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Nucleic Acid Electrophoresis:

Article Title: Lack of Glycoprotein 130/Signal Transducer and Activator of Transcription 3-Mediated Signaling in Hepatocytes Enhances Chronic Liver Injury and Fibrosis Progression in a Model of Sclerosing Cholangitis
Article Snippet: Paragraph title: SDS-Polyacrylamide Gel Electrophoresis and Western Blotting ... As a secondary antibody, horseradish peroxidase-linked anti-rabbit IgG (7074, Cell Signaling Technology) and horseradish peroxidase-linked anti-mouse IgG (sc-2005, Santa Cruz Biotechnology) were used.

Article Title: Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-?B and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines
Article Snippet: Aliquots of the cytosol fraction containing 50 μg of protein for ERK1/2 and JNK1/2 and 20 μg of protein for I-κBα were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10, 10, and 12% polyacrylamide gels, respectively. .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse immunoglobulin G (Santa Cruz Biotechnology) or rabbit polyclonal antibody against I-κBα (1:1,000 dilution) (Santa Cruz Biotechnology) for 1 h. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Blocking Assay:

Article Title: Antitumor effects and the underlying mechanism of licochalcone A combined with 5-fluorouracil in gastric cancer cells
Article Snippet: GAPDH antibodies were diluted to 1:5,000 in blocking solution. .. The secondary antibodies included horseradish peroxidase-linked anti-mouse immunoglobulin G (dilution, 1:5,000; sc-358914) and anti-rabbit immunoglobulin G (dilution, 1:4,000; sc-2357) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. The immunoreactive bands were visualized using the Enhanced Chemiluminescence plus Western Blotting Detection system, which was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

Real-time Polymerase Chain Reaction:

Article Title: Leptin modulates ? cell expression of IL-1 receptor antagonist and release of IL-1? in human islets
Article Snippet: Thereafter, membranes were incubated with horseradish-peroxidase-linked anti-mouse IgG (Santa Cruz Biotechnology) or streptavidin (Jackson ImmunoResearch). .. Total RNA extraction and quantitative PCR were performed as described ( ).

Incubation:

Article Title: Leptin modulates ? cell expression of IL-1 receptor antagonist and release of IL-1? in human islets
Article Snippet: .. Thereafter, membranes were incubated with horseradish-peroxidase-linked anti-mouse IgG (Santa Cruz Biotechnology) or streptavidin (Jackson ImmunoResearch). ..

Article Title: Lack of Glycoprotein 130/Signal Transducer and Activator of Transcription 3-Mediated Signaling in Hepatocytes Enhances Chronic Liver Injury and Fibrosis Progression in a Model of Sclerosing Cholangitis
Article Snippet: For primary antibody incubation, membranes were probed with anti-phosphorylated-STAT3 (Tyr-705, 9131s, Cell Signaling Technology, Danvers, MA), anti-STAT3 (STAT3, C-20, sc482, Santa Cruz Biotechnology, Heidelberg, Germany), and anti-GAPDH (4699-9555, Poole, Dorset, UK) antibodies. .. As a secondary antibody, horseradish peroxidase-linked anti-rabbit IgG (7074, Cell Signaling Technology) and horseradish peroxidase-linked anti-mouse IgG (sc-2005, Santa Cruz Biotechnology) were used.

Article Title: Differential Role of gp130-Dependent STAT and Ras Signalling for Haematopoiesis Following Bone-Marrow Transplantation
Article Snippet: SDS Page and Western Blot For primary antibody incubation, membranes were probed with anti-phospho-STAT-3 (Tyr705; #9131s; Cell Signalling, Frankfurt, Germany) and anti-GAPDH (4699–9555, Poole, Dorset, UK) antibodies. .. As a secondary antibody, HRP-linked anti-rabbit immunoglobulin G (#7074; Cell Signaling, Frankfurt, Germany) and HRP-linked anti-mouse immunoglobulin G (sc-2005, Santa Cruz, Heidelberg, Germany) were used.

Article Title: Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis
Article Snippet: .. After washing, the rabbit antibody stained blots were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3000, Cell Signaling), while the mouse antibody stained blots were incubated with HRP-linked anti-mouse IgG (1:6000, Santa Cruz) followed by washing and then detection with an ECL-Plus detection kit (Amersham, NJ). .. For experiments involving p-eIF2α, the lumbar spinal cord tissue from each mouse was collected and homogenized in 200μl homogenizing buffer (20mM Tris, pH 8.0, 150mM NaCl, 1mM EDTA, 0.5% Triton X-100, 0.5% SDS) containing a protease inhibitor cocktail.

Article Title: Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-?B and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines
Article Snippet: .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse immunoglobulin G (Santa Cruz Biotechnology) or rabbit polyclonal antibody against I-κBα (1:1,000 dilution) (Santa Cruz Biotechnology) for 1 h. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (Amersham Pharmacia Biotech). .. The relative densities of ERK1/2, JNK1/2, and I-κBα were determined by densitometric analysis, followed by photography of the specific bands (Kodak XLS-1 film).

Article Title: Moxifloxacin enhances antiproliferative and apoptotic effects of etoposide but inhibits its proinflammatory effects in THP-1 and Jurkat cells
Article Snippet: .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse IgG (Santa Cruz Biotechnology). .. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (ECL, Amersham Pharmacia Biotech, Uppsala, Sweden).

Article Title: Antitumor effects and the underlying mechanism of licochalcone A combined with 5-fluorouracil in gastric cancer cells
Article Snippet: Following blocking with 5% nonfat milk in blocking buffer (20 mM Tris-buffered saline containing 0.1% Tween-20; pH 7.5), the membranes were incubated with primary antibody at 4°C overnight and then incubated at room temperature for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody. .. The secondary antibodies included horseradish peroxidase-linked anti-mouse immunoglobulin G (dilution, 1:5,000; sc-358914) and anti-rabbit immunoglobulin G (dilution, 1:4,000; sc-2357) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. The immunoreactive bands were visualized using the Enhanced Chemiluminescence plus Western Blotting Detection system, which was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

BIA-KA:

Article Title: Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis
Article Snippet: The secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). .. Western-blotting detection regents, Glutathione-Sepharose 4B, Quikchange II site-directed mutagenesis Kit, and BCA Protein Assay kit were purchased from Amersham Biosciences (Piscataway, NJ, USA).

Western Blot:

Article Title: Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis
Article Snippet: The secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). .. Western-blotting detection regents, Glutathione-Sepharose 4B, Quikchange II site-directed mutagenesis Kit, and BCA Protein Assay kit were purchased from Amersham Biosciences (Piscataway, NJ, USA).

Article Title: Lack of Glycoprotein 130/Signal Transducer and Activator of Transcription 3-Mediated Signaling in Hepatocytes Enhances Chronic Liver Injury and Fibrosis Progression in a Model of Sclerosing Cholangitis
Article Snippet: Paragraph title: SDS-Polyacrylamide Gel Electrophoresis and Western Blotting ... As a secondary antibody, horseradish peroxidase-linked anti-rabbit IgG (7074, Cell Signaling Technology) and horseradish peroxidase-linked anti-mouse IgG (sc-2005, Santa Cruz Biotechnology) were used.

Article Title: Differential Role of gp130-Dependent STAT and Ras Signalling for Haematopoiesis Following Bone-Marrow Transplantation
Article Snippet: Paragraph title: SDS Page and Western Blot ... As a secondary antibody, HRP-linked anti-rabbit immunoglobulin G (#7074; Cell Signaling, Frankfurt, Germany) and HRP-linked anti-mouse immunoglobulin G (sc-2005, Santa Cruz, Heidelberg, Germany) were used.

Article Title: Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis
Article Snippet: Paragraph title: Western blot ... After washing, the rabbit antibody stained blots were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3000, Cell Signaling), while the mouse antibody stained blots were incubated with HRP-linked anti-mouse IgG (1:6000, Santa Cruz) followed by washing and then detection with an ECL-Plus detection kit (Amersham, NJ).

Article Title: Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH
Article Snippet: Paragraph title: 2.5. Western Blot Analyses ... The following secondary antibodies have been used in this study: HRP-linked anti-rabbit immunoglobulin G (7074, Cell Signaling) and HRP-linked anti-mouse immunoglobulin G (sc-2005, Santa Cruz).

Article Title: Therapeutic intervention of silymarin on the migration of non-small cell lung cancer cells is associated with the axis of multiple molecular targets including class 1 HDACs, ZEB1 expression, and restoration of miR-203 and E-cadherin expression
Article Snippet: Purified silymarin ( > 98%) was purchased from Sigma Chemical Co. Primary antibodies were purchased as follows: antibodies for class I HDAC (anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8); anti-histone H3; ZEB1; E-cadherin; β-actin; and secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G, were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). .. The enhanced chemiluminescence western blotting detection reagents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Article Title: Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-?B and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines
Article Snippet: Paragraph title: WB analysis of ERK, I-κBα, and JNK. ... The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse immunoglobulin G (Santa Cruz Biotechnology) or rabbit polyclonal antibody against I-κBα (1:1,000 dilution) (Santa Cruz Biotechnology) for 1 h. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Article Title: Moxifloxacin enhances antiproliferative and apoptotic effects of etoposide but inhibits its proinflammatory effects in THP-1 and Jurkat cells
Article Snippet: Paragraph title: Western blot analysis of caspase-3 ... The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse IgG (Santa Cruz Biotechnology).

Article Title: Antitumor effects and the underlying mechanism of licochalcone A combined with 5-fluorouracil in gastric cancer cells
Article Snippet: .. The secondary antibodies included horseradish peroxidase-linked anti-mouse immunoglobulin G (dilution, 1:5,000; sc-358914) and anti-rabbit immunoglobulin G (dilution, 1:4,000; sc-2357) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. The immunoreactive bands were visualized using the Enhanced Chemiluminescence plus Western Blotting Detection system, which was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). ..

Transfection:

Article Title: Therapeutic intervention of silymarin on the migration of non-small cell lung cancer cells is associated with the axis of multiple molecular targets including class 1 HDACs, ZEB1 expression, and restoration of miR-203 and E-cadherin expression
Article Snippet: Purified silymarin ( > 98%) was purchased from Sigma Chemical Co. Primary antibodies were purchased as follows: antibodies for class I HDAC (anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8); anti-histone H3; ZEB1; E-cadherin; β-actin; and secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G, were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). .. ZEB1 siRNA transfection kit and transfection reagents were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz).

Protease Inhibitor:

Article Title: Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis
Article Snippet: After washing, the rabbit antibody stained blots were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3000, Cell Signaling), while the mouse antibody stained blots were incubated with HRP-linked anti-mouse IgG (1:6000, Santa Cruz) followed by washing and then detection with an ECL-Plus detection kit (Amersham, NJ). .. For experiments involving p-eIF2α, the lumbar spinal cord tissue from each mouse was collected and homogenized in 200μl homogenizing buffer (20mM Tris, pH 8.0, 150mM NaCl, 1mM EDTA, 0.5% Triton X-100, 0.5% SDS) containing a protease inhibitor cocktail.

Article Title: Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-?B and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines
Article Snippet: For the analysis of JNK, after incubation for 0 to 120 min, the cells were collected on ice, washed twice with ice-cold PBS, and suspended in 80 μl of the homogenization buffer: 40 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM EDTA (pH 8.0), 0.1 mM Na3 VO4 , 50 mM β-glycerophosphate, 1 mM benzamidine, 1 mM dithiothreitol (DTT), and 1:100 Complete Mini Protease Inhibitor cocktail (Boehringer Mannheim). .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse immunoglobulin G (Santa Cruz Biotechnology) or rabbit polyclonal antibody against I-κBα (1:1,000 dilution) (Santa Cruz Biotechnology) for 1 h. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Cell Culture:

Article Title: Leptin modulates ? cell expression of IL-1 receptor antagonist and release of IL-1? in human islets
Article Snippet: Cultured human islets were fixed in 2.5% paraformaldehyde/0.1% glutaraldehyde/0.01% picric acid and dehydrated in graded ethanol before embedding in LR white (Polysciences). .. Thereafter, membranes were incubated with horseradish-peroxidase-linked anti-mouse IgG (Santa Cruz Biotechnology) or streptavidin (Jackson ImmunoResearch).

Sonication:

Article Title: Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-?B and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines
Article Snippet: After incubation overnight at −70°C, the lysates were sonicated (3 s on and 1 s off; total time, 6 min) and then subjected to centrifugation (20,000 × g ) at 4°C for 15 min to obtain a cytosolic fraction. .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse immunoglobulin G (Santa Cruz Biotechnology) or rabbit polyclonal antibody against I-κBα (1:1,000 dilution) (Santa Cruz Biotechnology) for 1 h. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Affinity Purification:

Article Title: N,N'-Dinitrosopiperazine-Mediated Heat-Shock Protein 70-2 Expression Is Involved in Metastasis of Nasopharyngeal Carcinoma
Article Snippet: The HSP70t polyclonal antibody was affinity purified from rabbit and raised against a synthetic HSP70tL peptide of human origin. .. Secondary antibodies used in these experiments were horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G and purchased from Santa Cruz Biotechnology, Inc. Immunoblotting detection regents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Molecular Weight:

Article Title: Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis
Article Snippet: A human-specific rabbit anti-SOD1 antibody was used to detect both monomeric and high molecular weight forms of mtSOD1, and anti-β-tubulin antibody (1:500, Developmental Studies Hybridoma Bank) was used to stain for β-tubulin as a control for the amount of protein loaded. (ii) The homogenates were diluted 1:5 (2μg of total protein) followed by boiling in loading buffer that contained β-mercaptoethanol and then loaded on to 15% SDS polyacrylamide gels for western blot analysis. .. After washing, the rabbit antibody stained blots were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3000, Cell Signaling), while the mouse antibody stained blots were incubated with HRP-linked anti-mouse IgG (1:6000, Santa Cruz) followed by washing and then detection with an ECL-Plus detection kit (Amersham, NJ).

MTT Assay:

Article Title: N,N'-Dinitrosopiperazine-Mediated Heat-Shock Protein 70-2 Expression Is Involved in Metastasis of Nasopharyngeal Carcinoma
Article Snippet: Secondary antibodies used in these experiments were horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G and purchased from Santa Cruz Biotechnology, Inc. Immunoblotting detection regents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). .. Dimethyl sulfoxide (DMSO), fluorescein isothiocyanate (FITC) antibody, 4′,6-diamidino-2-phenylindole (DAPI), and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)- 2-(4-ulfophenyl)-2H-tetrazolium (MTT), were purchased from Sigma-Aldrich.

HDAC Activity Assay:

Article Title: Therapeutic intervention of silymarin on the migration of non-small cell lung cancer cells is associated with the axis of multiple molecular targets including class 1 HDACs, ZEB1 expression, and restoration of miR-203 and E-cadherin expression
Article Snippet: Purified silymarin ( > 98%) was purchased from Sigma Chemical Co. Primary antibodies were purchased as follows: antibodies for class I HDAC (anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8); anti-histone H3; ZEB1; E-cadherin; β-actin; and secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G, were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). .. The HDAC Activity Assay Kit was purchased from Active Motif (Catalog # 56210, Carlsbad, CA).

Mutagenesis:

Article Title: Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis
Article Snippet: The secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). .. Western-blotting detection regents, Glutathione-Sepharose 4B, Quikchange II site-directed mutagenesis Kit, and BCA Protein Assay kit were purchased from Amersham Biosciences (Piscataway, NJ, USA).

Purification:

Article Title: Therapeutic intervention of silymarin on the migration of non-small cell lung cancer cells is associated with the axis of multiple molecular targets including class 1 HDACs, ZEB1 expression, and restoration of miR-203 and E-cadherin expression
Article Snippet: .. Purified silymarin ( > 98%) was purchased from Sigma Chemical Co. Primary antibodies were purchased as follows: antibodies for class I HDAC (anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8); anti-histone H3; ZEB1; E-cadherin; β-actin; and secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G, were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). .. The HDAC Activity Assay Kit was purchased from Active Motif (Catalog # 56210, Carlsbad, CA).

Immunostaining:

Article Title: Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis
Article Snippet: After washing, the rabbit antibody stained blots were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3000, Cell Signaling), while the mouse antibody stained blots were incubated with HRP-linked anti-mouse IgG (1:6000, Santa Cruz) followed by washing and then detection with an ECL-Plus detection kit (Amersham, NJ). .. Immunostaining was carried out with overnight incubation at 4°C with anti-rabbit p-eIF2α antibody (1:1,000, cat#.3597, Cell Signaling Technology) and mouse β-tubulin serum (to stain for β-tubulin as a control for the amount of protein loaded).

Chloramphenicol Acetyltransferase Assay:

Article Title: Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis
Article Snippet: After washing, the rabbit antibody stained blots were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3000, Cell Signaling), while the mouse antibody stained blots were incubated with HRP-linked anti-mouse IgG (1:6000, Santa Cruz) followed by washing and then detection with an ECL-Plus detection kit (Amersham, NJ). .. Immunostaining was carried out with overnight incubation at 4°C with anti-rabbit p-eIF2α antibody (1:1,000, cat.3597, Cell Signaling Technology) and mouse β-tubulin serum (to stain for β-tubulin as a control for the amount of protein loaded).

SDS Page:

Article Title: Differential Role of gp130-Dependent STAT and Ras Signalling for Haematopoiesis Following Bone-Marrow Transplantation
Article Snippet: Paragraph title: SDS Page and Western Blot ... As a secondary antibody, HRP-linked anti-rabbit immunoglobulin G (#7074; Cell Signaling, Frankfurt, Germany) and HRP-linked anti-mouse immunoglobulin G (sc-2005, Santa Cruz, Heidelberg, Germany) were used.

Article Title: Antitumor effects and the underlying mechanism of licochalcone A combined with 5-fluorouracil in gastric cancer cells
Article Snippet: The proteins (50 µg) were separated using 5–10% SDS-PAGE and transferred onto a nitrocellulose membrane. .. The secondary antibodies included horseradish peroxidase-linked anti-mouse immunoglobulin G (dilution, 1:5,000; sc-358914) and anti-rabbit immunoglobulin G (dilution, 1:4,000; sc-2357) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. The immunoreactive bands were visualized using the Enhanced Chemiluminescence plus Western Blotting Detection system, which was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

Software:

Article Title: Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH
Article Snippet: The following secondary antibodies have been used in this study: HRP-linked anti-rabbit immunoglobulin G (7074, Cell Signaling) and HRP-linked anti-mouse immunoglobulin G (sc-2005, Santa Cruz). .. Enhanced chemiluminescence (ECL) method was used to detect protein bands and the software ImageJ was employed for densitometric analysis of band intensity.

Electrophoresis:

Article Title: Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH
Article Snippet: Upon electrophoresis and transfer blotting, Ponceau-Red staining was used to verify transfer efficiency and equal protein loading. .. The following secondary antibodies have been used in this study: HRP-linked anti-rabbit immunoglobulin G (7074, Cell Signaling) and HRP-linked anti-mouse immunoglobulin G (sc-2005, Santa Cruz).

Article Title: Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-?B and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines
Article Snippet: After electrophoresis and electrophoretic transfer of the proteins to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), the membranes were blocked with 3% nonfat milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween (TBST) for 1 h. The membranes were then rinsed three times in TBST and incubated at room temperature (RT) with mouse monoclonal anti-MAPK antibody and with activated, diphosphorylated, ERK-1/2, phospho-p44/42 MAPK antibody (Sigma Chemical Co.), and anti-phospho-JNK1/2, phospho-p46/54 JNK antibody (New England Biolabs, Beverly, Mass.) (1:20,000 and 1:1,000 dilutions, respectively) and anti-ERK-1/2 antibody and anti-JNK1/2 antibody (1:1,000 dilution) or rabbit polyclonal antibody against I-κB (1:1,000 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) for 1 h. Actin levels were also assessed as a loading control by using an antibody (Santa Cruz Biotechnology) that reacts with a broad range of actin isoforms. .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse immunoglobulin G (Santa Cruz Biotechnology) or rabbit polyclonal antibody against I-κBα (1:1,000 dilution) (Santa Cruz Biotechnology) for 1 h. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Article Title: Moxifloxacin enhances antiproliferative and apoptotic effects of etoposide but inhibits its proinflammatory effects in THP-1 and Jurkat cells
Article Snippet: Equal amounts of protein (50 μ g) were subjected to electrophoresis on 12% SDS–polyacrylamide gel and then electrophoretically transferred to a nitrocellulose membrane. .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse IgG (Santa Cruz Biotechnology).

Homogenization:

Article Title: Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-?B and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines
Article Snippet: For the analysis of JNK, after incubation for 0 to 120 min, the cells were collected on ice, washed twice with ice-cold PBS, and suspended in 80 μl of the homogenization buffer: 40 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM EDTA (pH 8.0), 0.1 mM Na3 VO4 , 50 mM β-glycerophosphate, 1 mM benzamidine, 1 mM dithiothreitol (DTT), and 1:100 Complete Mini Protease Inhibitor cocktail (Boehringer Mannheim). .. The blots were then incubated with a secondary antibody, horseradish peroxidase-linked anti-mouse immunoglobulin G (Santa Cruz Biotechnology) or rabbit polyclonal antibody against I-κBα (1:1,000 dilution) (Santa Cruz Biotechnology) for 1 h. After 1 h at RT and three washes in TBST, the blots were incubated in enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Concentration Assay:

Article Title: Berberine Inhibits Metastasis of Nasopharyngeal Carcinoma 5-8F Cells by Targeting Rho Kinase-mediated Ezrin Phosphorylation at Threonine 567 *
Article Snippet: The final DMSO concentration in the medium applied to cells was 0.1% (in both control and treated groups) without affecting cell viability. .. The secondary antibodies, horseradish peroxidase-linked anti-mouse IgG and anti-rabbit IgG, were purchased from Santa Cruz Biotechnology, Inc. GST-Rhotekin-RBD protein-agarose beads were purchased from Cytoskeleton Inc. (Denver, CO).

Staining:

Article Title: Lack of Glycoprotein 130/Signal Transducer and Activator of Transcription 3-Mediated Signaling in Hepatocytes Enhances Chronic Liver Injury and Fibrosis Progression in a Model of Sclerosing Cholangitis
Article Snippet: Ponceau Red staining was used to demonstrate equal protein loading. .. As a secondary antibody, horseradish peroxidase-linked anti-rabbit IgG (7074, Cell Signaling Technology) and horseradish peroxidase-linked anti-mouse IgG (sc-2005, Santa Cruz Biotechnology) were used.

Article Title: Guanabenz, which enhances the unfolded protein response, ameliorates mutant SOD1-induced amyotrophic lateral sclerosis
Article Snippet: .. After washing, the rabbit antibody stained blots were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3000, Cell Signaling), while the mouse antibody stained blots were incubated with HRP-linked anti-mouse IgG (1:6000, Santa Cruz) followed by washing and then detection with an ECL-Plus detection kit (Amersham, NJ). .. For experiments involving p-eIF2α, the lumbar spinal cord tissue from each mouse was collected and homogenized in 200μl homogenizing buffer (20mM Tris, pH 8.0, 150mM NaCl, 1mM EDTA, 0.5% Triton X-100, 0.5% SDS) containing a protease inhibitor cocktail.

Article Title: Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH
Article Snippet: Upon electrophoresis and transfer blotting, Ponceau-Red staining was used to verify transfer efficiency and equal protein loading. .. The following secondary antibodies have been used in this study: HRP-linked anti-rabbit immunoglobulin G (7074, Cell Signaling) and HRP-linked anti-mouse immunoglobulin G (sc-2005, Santa Cruz).

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  • 82
    Santa Cruz Biotechnology hrp linked mouse monoclonal igg his probe antibody h 3
    Functional significance of trimeric contacts in CaiT. (a) Western blot analysis of CaiT with given amino acid replacements in E. coli JW0039 membranes. <t>HRP-linked</t> mouse anti-His <t>IgG</t> was used to detect CaiT. PageRuler Prestained Protein Ladder was used for molecular size estimation. Shown is the section of the blot that contains the CaiT band. The complete gel is presented in Fig. S6. (b) Initial rates of counterflow (black) and maximum accumulation of carnitine in E. coli JW0039 (white) were determined as described in the legend of Fig. 3 . As negative control, cells carrying pET21a ( nc ) were used. (c) Initial rates of counterflow (black) and maximum accumulation (white) of carnitine catalyzed by CaiT variants in proteoliposomes. CaiT was purified and reconstituted as described 1 . The lipid to protein ratio of the resulting proteoliposomes was 100:1 (w/w). Proteoliposomes were preloaded with 10 mM unlabeled L-carnitine overnight. Aliquots of the proteoliposome suspension (1–2 µg of protein) were then diluted into 400 µL buffer containing 4.5 µM L-[methyl- 14 C]carnitine (55 Ci/mol) resulting in a final carnitine concentration of 54.5 µM. As negative control, proteoliposomes not preloaded with unlabeled carnitine ( nc ) were used and the obtained transport rates were used for correcting the data. Carnitine counterflow with cells or proteoliposomes was assayed at 25 °C under aerobic conditions using a rapid filtration method as described 1 . (d) Michaelis-Menten kinetics of L-carnitine counterflow in proteoliposomes reconstituted with wild-type CaiT (WT) or CaiT-D288A. Initial rates of L-[methyl- 14 C]carnitine uptake were measured at given substrate concentrations and plotted using the kinetic module of GraphPad Prism. For all experiments, standard deviations were calculated from triplicate determinations of a representative experiment.
    Hrp Linked Mouse Monoclonal Igg His Probe Antibody H 3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp linked mouse monoclonal igg his probe antibody h 3/product/Santa Cruz Biotechnology
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    89
    Santa Cruz Biotechnology horseradish peroxidase linked anti mouse immunoglobulin g
    Phospho-ezrin and ezrin expression in NPC metastatic nodes. Phospho-ezrin and ezrin were detected in the primary and metastatic NPC samples using immunohistochemistry. ( a , b ) primary NPC and metastatic tumor sections were stained with hematoxylin and eosin; ( c , d ) stained with antibodies against ezrin; ( e , f ) stained with phospho-ezrin at Thr567; ( g , h ) stained with <t>IgG,</t> served as a blank control. Arrow, positive cells. Original magnification, ×400. Scale bar, 5 μm. H E, hematoxylin and eosin.
    Horseradish Peroxidase Linked Anti Mouse Immunoglobulin G, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase linked anti mouse immunoglobulin g/product/Santa Cruz Biotechnology
    Average 89 stars, based on 1 article reviews
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    78
    Santa Cruz Biotechnology anti mouse horseradish peroxidase linked immunoglobulin g igg
    Microfilament inhibitors suppress the AngII-mediated increase in COX-2 expression by preventing HuR binding to the corresponding mRNA. ( A ). HMC cells were treated for 2 h with either vehicle (−) or with AngII, or together with different microfilament inhibitors as indicated. Inhibitors were preincubated for 30 min (Lat.A) or 4 h (Blebbistat.) before the addition of AngII. To achieve transcriptional induction of different HuR target genes, cells were stimulated for 16 h with a cytokine mix containing IL-1β and TNFα (both at 2 nM) before AngII treatment. Steady-state mRNA levels of COX-2, cyclin A and cyclin D 1 were quantified by qRT-PCR using β-actin as a normalization control. Results are depicted as relative mRNA levels (%) in comparison with cytokine-stimulated conditions (vehicle), which were set to 100% and are means ± SD (n=3). ** P ≤ 0.01, *** P ≤ 0.005 compared with cytokine-induced conditions ## P ≤ 0.01 and ### P ≤ 0.005, compared with cytokine plus AngII-treated conditions. ( B ). Reduction of AngII-mediated stabilization of cytokine-triggered COX- 2 mRNA by blebbistatin (left panel) or latrunculin A (right panel). Quiescent HMC were treated for 20 h with a cytokine mixture containing IL-1β and TNFα (both at 2 nM) before the administration of Act D, which was added 30 min before AngII (filled triangles). Importantly, the indicated cytoskeletal inhibitors were given 4.5 h before the addition of AngII and remained for the indicated times with vehicle (open circles) or with 0.1 µM AngII (open squares) before cells were harvested and extracted for total cellular RNA. Steady-state mRNA levels of COX-2 were determined by qRT-PCR using β-actin mRNA as a normalization control. Graphs show means ± SD (n = 3) and depict the percentage of remaining COX-2 mRNA levels compared with the content of steady-state COX-2 mRNA measured immediately before the addition of Act D (0 h). ( C ). HMC were treated similarly as described in (A) before total cell lysates were assayed by IP using an anti-HuR antibody (gray bars), or the same amount of isotypic <t>IgG</t> (open bars). HuR-bound COX-2 mRNA was determined by pulldown RT assays. Data shown are means of two independent experiments and are depicted as fold induction versus cytokine-treated cells.
    Anti Mouse Horseradish Peroxidase Linked Immunoglobulin G Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti mouse horseradish peroxidase linked immunoglobulin g igg - by Bioz Stars, 2020-02
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    Image Search Results


    Functional significance of trimeric contacts in CaiT. (a) Western blot analysis of CaiT with given amino acid replacements in E. coli JW0039 membranes. HRP-linked mouse anti-His IgG was used to detect CaiT. PageRuler Prestained Protein Ladder was used for molecular size estimation. Shown is the section of the blot that contains the CaiT band. The complete gel is presented in Fig. S6. (b) Initial rates of counterflow (black) and maximum accumulation of carnitine in E. coli JW0039 (white) were determined as described in the legend of Fig. 3 . As negative control, cells carrying pET21a ( nc ) were used. (c) Initial rates of counterflow (black) and maximum accumulation (white) of carnitine catalyzed by CaiT variants in proteoliposomes. CaiT was purified and reconstituted as described 1 . The lipid to protein ratio of the resulting proteoliposomes was 100:1 (w/w). Proteoliposomes were preloaded with 10 mM unlabeled L-carnitine overnight. Aliquots of the proteoliposome suspension (1–2 µg of protein) were then diluted into 400 µL buffer containing 4.5 µM L-[methyl- 14 C]carnitine (55 Ci/mol) resulting in a final carnitine concentration of 54.5 µM. As negative control, proteoliposomes not preloaded with unlabeled carnitine ( nc ) were used and the obtained transport rates were used for correcting the data. Carnitine counterflow with cells or proteoliposomes was assayed at 25 °C under aerobic conditions using a rapid filtration method as described 1 . (d) Michaelis-Menten kinetics of L-carnitine counterflow in proteoliposomes reconstituted with wild-type CaiT (WT) or CaiT-D288A. Initial rates of L-[methyl- 14 C]carnitine uptake were measured at given substrate concentrations and plotted using the kinetic module of GraphPad Prism. For all experiments, standard deviations were calculated from triplicate determinations of a representative experiment.

    Journal: Scientific Reports

    Article Title: Comparison of the functional properties of trimeric and monomeric CaiT of Escherichia coli

    doi: 10.1038/s41598-019-40516-7

    Figure Lengend Snippet: Functional significance of trimeric contacts in CaiT. (a) Western blot analysis of CaiT with given amino acid replacements in E. coli JW0039 membranes. HRP-linked mouse anti-His IgG was used to detect CaiT. PageRuler Prestained Protein Ladder was used for molecular size estimation. Shown is the section of the blot that contains the CaiT band. The complete gel is presented in Fig. S6. (b) Initial rates of counterflow (black) and maximum accumulation of carnitine in E. coli JW0039 (white) were determined as described in the legend of Fig. 3 . As negative control, cells carrying pET21a ( nc ) were used. (c) Initial rates of counterflow (black) and maximum accumulation (white) of carnitine catalyzed by CaiT variants in proteoliposomes. CaiT was purified and reconstituted as described 1 . The lipid to protein ratio of the resulting proteoliposomes was 100:1 (w/w). Proteoliposomes were preloaded with 10 mM unlabeled L-carnitine overnight. Aliquots of the proteoliposome suspension (1–2 µg of protein) were then diluted into 400 µL buffer containing 4.5 µM L-[methyl- 14 C]carnitine (55 Ci/mol) resulting in a final carnitine concentration of 54.5 µM. As negative control, proteoliposomes not preloaded with unlabeled carnitine ( nc ) were used and the obtained transport rates were used for correcting the data. Carnitine counterflow with cells or proteoliposomes was assayed at 25 °C under aerobic conditions using a rapid filtration method as described 1 . (d) Michaelis-Menten kinetics of L-carnitine counterflow in proteoliposomes reconstituted with wild-type CaiT (WT) or CaiT-D288A. Initial rates of L-[methyl- 14 C]carnitine uptake were measured at given substrate concentrations and plotted using the kinetic module of GraphPad Prism. For all experiments, standard deviations were calculated from triplicate determinations of a representative experiment.

    Article Snippet: Relative amounts of CaiT with given amino acid replacements in membranes of E. coli JW0039 harboring plasmid pT7-5/caiT were estimated by Western blot analysis with HRP-linked mouse monoclonal IgG His-probe Antibody (H-3) (Santa Cruz Biotechnology) directed against the His tag at the C terminus of each CaiT variant by the enhances chemiluminescence method according to the manufacturer’s protocol.

    Techniques: Functional Assay, Western Blot, Negative Control, Purification, Concentration Assay, Filtration

    Phospho-ezrin and ezrin expression in NPC metastatic nodes. Phospho-ezrin and ezrin were detected in the primary and metastatic NPC samples using immunohistochemistry. ( a , b ) primary NPC and metastatic tumor sections were stained with hematoxylin and eosin; ( c , d ) stained with antibodies against ezrin; ( e , f ) stained with phospho-ezrin at Thr567; ( g , h ) stained with IgG, served as a blank control. Arrow, positive cells. Original magnification, ×400. Scale bar, 5 μm. H E, hematoxylin and eosin.

    Journal: International Journal of Molecular Sciences

    Article Title: Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis

    doi: 10.3390/ijms151120054

    Figure Lengend Snippet: Phospho-ezrin and ezrin expression in NPC metastatic nodes. Phospho-ezrin and ezrin were detected in the primary and metastatic NPC samples using immunohistochemistry. ( a , b ) primary NPC and metastatic tumor sections were stained with hematoxylin and eosin; ( c , d ) stained with antibodies against ezrin; ( e , f ) stained with phospho-ezrin at Thr567; ( g , h ) stained with IgG, served as a blank control. Arrow, positive cells. Original magnification, ×400. Scale bar, 5 μm. H E, hematoxylin and eosin.

    Article Snippet: The secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Expressing, Immunohistochemistry, Staining

    Validation of the representative phosphoproteins by Western-blotting. Representative proteins ezrin ( A ); vimentin ( B ); stathmin ( C ); STAT3 ( D ) of the immunoprecipitated proteins were detected by Western-blotting using anti-serine or threonine phosphorylation antibody. IgG served as the control. The relative Western-blotting ratio of phosphorylation levels of every protein was normalized to its corresponding proteomic data.

    Journal: International Journal of Molecular Sciences

    Article Title: Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis

    doi: 10.3390/ijms151120054

    Figure Lengend Snippet: Validation of the representative phosphoproteins by Western-blotting. Representative proteins ezrin ( A ); vimentin ( B ); stathmin ( C ); STAT3 ( D ) of the immunoprecipitated proteins were detected by Western-blotting using anti-serine or threonine phosphorylation antibody. IgG served as the control. The relative Western-blotting ratio of phosphorylation levels of every protein was normalized to its corresponding proteomic data.

    Article Snippet: The secondary antibodies, horseradish peroxidase-linked anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Western Blot, Immunoprecipitation

    Microfilament inhibitors suppress the AngII-mediated increase in COX-2 expression by preventing HuR binding to the corresponding mRNA. ( A ). HMC cells were treated for 2 h with either vehicle (−) or with AngII, or together with different microfilament inhibitors as indicated. Inhibitors were preincubated for 30 min (Lat.A) or 4 h (Blebbistat.) before the addition of AngII. To achieve transcriptional induction of different HuR target genes, cells were stimulated for 16 h with a cytokine mix containing IL-1β and TNFα (both at 2 nM) before AngII treatment. Steady-state mRNA levels of COX-2, cyclin A and cyclin D 1 were quantified by qRT-PCR using β-actin as a normalization control. Results are depicted as relative mRNA levels (%) in comparison with cytokine-stimulated conditions (vehicle), which were set to 100% and are means ± SD (n=3). ** P ≤ 0.01, *** P ≤ 0.005 compared with cytokine-induced conditions ## P ≤ 0.01 and ### P ≤ 0.005, compared with cytokine plus AngII-treated conditions. ( B ). Reduction of AngII-mediated stabilization of cytokine-triggered COX- 2 mRNA by blebbistatin (left panel) or latrunculin A (right panel). Quiescent HMC were treated for 20 h with a cytokine mixture containing IL-1β and TNFα (both at 2 nM) before the administration of Act D, which was added 30 min before AngII (filled triangles). Importantly, the indicated cytoskeletal inhibitors were given 4.5 h before the addition of AngII and remained for the indicated times with vehicle (open circles) or with 0.1 µM AngII (open squares) before cells were harvested and extracted for total cellular RNA. Steady-state mRNA levels of COX-2 were determined by qRT-PCR using β-actin mRNA as a normalization control. Graphs show means ± SD (n = 3) and depict the percentage of remaining COX-2 mRNA levels compared with the content of steady-state COX-2 mRNA measured immediately before the addition of Act D (0 h). ( C ). HMC were treated similarly as described in (A) before total cell lysates were assayed by IP using an anti-HuR antibody (gray bars), or the same amount of isotypic IgG (open bars). HuR-bound COX-2 mRNA was determined by pulldown RT assays. Data shown are means of two independent experiments and are depicted as fold induction versus cytokine-treated cells.

    Journal: Nucleic Acids Research

    Article Title: RNA-dependent association with myosin IIA promotes F-actin-guided trafficking of the ELAV-like protein HuR to polysomes

    doi: 10.1093/nar/gkt663

    Figure Lengend Snippet: Microfilament inhibitors suppress the AngII-mediated increase in COX-2 expression by preventing HuR binding to the corresponding mRNA. ( A ). HMC cells were treated for 2 h with either vehicle (−) or with AngII, or together with different microfilament inhibitors as indicated. Inhibitors were preincubated for 30 min (Lat.A) or 4 h (Blebbistat.) before the addition of AngII. To achieve transcriptional induction of different HuR target genes, cells were stimulated for 16 h with a cytokine mix containing IL-1β and TNFα (both at 2 nM) before AngII treatment. Steady-state mRNA levels of COX-2, cyclin A and cyclin D 1 were quantified by qRT-PCR using β-actin as a normalization control. Results are depicted as relative mRNA levels (%) in comparison with cytokine-stimulated conditions (vehicle), which were set to 100% and are means ± SD (n=3). ** P ≤ 0.01, *** P ≤ 0.005 compared with cytokine-induced conditions ## P ≤ 0.01 and ### P ≤ 0.005, compared with cytokine plus AngII-treated conditions. ( B ). Reduction of AngII-mediated stabilization of cytokine-triggered COX- 2 mRNA by blebbistatin (left panel) or latrunculin A (right panel). Quiescent HMC were treated for 20 h with a cytokine mixture containing IL-1β and TNFα (both at 2 nM) before the administration of Act D, which was added 30 min before AngII (filled triangles). Importantly, the indicated cytoskeletal inhibitors were given 4.5 h before the addition of AngII and remained for the indicated times with vehicle (open circles) or with 0.1 µM AngII (open squares) before cells were harvested and extracted for total cellular RNA. Steady-state mRNA levels of COX-2 were determined by qRT-PCR using β-actin mRNA as a normalization control. Graphs show means ± SD (n = 3) and depict the percentage of remaining COX-2 mRNA levels compared with the content of steady-state COX-2 mRNA measured immediately before the addition of Act D (0 h). ( C ). HMC were treated similarly as described in (A) before total cell lysates were assayed by IP using an anti-HuR antibody (gray bars), or the same amount of isotypic IgG (open bars). HuR-bound COX-2 mRNA was determined by pulldown RT assays. Data shown are means of two independent experiments and are depicted as fold induction versus cytokine-treated cells.

    Article Snippet: Antibodies raised against HuR, histone deacetylase-1 (HDAC1), c-myc, anti-rabbit and anti-mouse horseradish peroxidase linked immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Activated Clotting Time Assay

    AngII promotes a physical interaction of HuR with non-muscle myosin IIA, which structurally depends on phosphorylated S318 within the RRM3. ( A , upper panel). Schematic representation of c-myc-tagged wild-type HuR and different deletion mutants bearing truncations in the specific HuR domains. (A, lower panel). HMC were transfected with the indicated c-myc-HuR coding plasmids. Seventy-two hours after transfection, cells were treated without (−) or with AngII (+) for 2 h. Thereafter, c-myc-tagged HuR proteins were immunoprecipitated with an anti-c-myc antibody (IP: c-myc), or isotype specific control (IP: IgG). HuR-bound myosin IIA was detected by Western blot analysis with an anti-myosin IIA specific antibody. Equal pull-down (input levels) of c-myc was ascertained by reincubating the blot with the same antibody used for IP. ( B ). Blot-overlay assay demonstrating that the hinge region of HuR is not relevant for AngII-induced myosin IIA binding. Pull-down of myosin IIA from vehicle (−) or AngII treated HMC was performed as described in ‘Materials and Methods’ section . Blots were probed with equal amounts of total cell lysates cells overexpressing c-myc-HuR wild-type or c-myc-HuRΔHinge. Myosin IIA-HuR interaction was assessed by reincubating the blot with an anti-c-myc antibody. Equal pull-down of myosin is ascertained by reincubating the blot with the same antibody used for the myosin IIA co-IP. ( C ). HMC were transfected with the indicated c-myc-point mutated HuR and IPs were performed similar as described for (A). Equal pull-down (input levels) of c-myc was ascertained by reincubating the blot with the same antibody used for IP. ( D , left panel). HuR binding to myosin IIA is sensitive toward RNase treatment. HMC overexpressing c-myc-HuRwild-type or c-myc-HuRΔHinge were treated for 2 h with AngII (0.1 µM) before total cell lysates from transfectants were immunoprecipitated with an anti-c-myc antibody or with control IgG. Before the IP reaction, the cell homogenates were subjected to RNase treatment and subsequently immunoblotted with an anti-myosin IIA specific antibody. Equal pull-down of c-myc is ascertained by reincubating the blot with anti-c-myc antiserum. (right panel). To test for AngII-dependent association of myosinIIA with endogenous HuR, 200 µg of total cell extracts from unstimulated (−) or AngII-stimulated (+) HMC were subjected to (IP) as described in ‘Materials and Methods’ section. The data shown are from a single experiment representative of two repeats with similar results.

    Journal: Nucleic Acids Research

    Article Title: RNA-dependent association with myosin IIA promotes F-actin-guided trafficking of the ELAV-like protein HuR to polysomes

    doi: 10.1093/nar/gkt663

    Figure Lengend Snippet: AngII promotes a physical interaction of HuR with non-muscle myosin IIA, which structurally depends on phosphorylated S318 within the RRM3. ( A , upper panel). Schematic representation of c-myc-tagged wild-type HuR and different deletion mutants bearing truncations in the specific HuR domains. (A, lower panel). HMC were transfected with the indicated c-myc-HuR coding plasmids. Seventy-two hours after transfection, cells were treated without (−) or with AngII (+) for 2 h. Thereafter, c-myc-tagged HuR proteins were immunoprecipitated with an anti-c-myc antibody (IP: c-myc), or isotype specific control (IP: IgG). HuR-bound myosin IIA was detected by Western blot analysis with an anti-myosin IIA specific antibody. Equal pull-down (input levels) of c-myc was ascertained by reincubating the blot with the same antibody used for IP. ( B ). Blot-overlay assay demonstrating that the hinge region of HuR is not relevant for AngII-induced myosin IIA binding. Pull-down of myosin IIA from vehicle (−) or AngII treated HMC was performed as described in ‘Materials and Methods’ section . Blots were probed with equal amounts of total cell lysates cells overexpressing c-myc-HuR wild-type or c-myc-HuRΔHinge. Myosin IIA-HuR interaction was assessed by reincubating the blot with an anti-c-myc antibody. Equal pull-down of myosin is ascertained by reincubating the blot with the same antibody used for the myosin IIA co-IP. ( C ). HMC were transfected with the indicated c-myc-point mutated HuR and IPs were performed similar as described for (A). Equal pull-down (input levels) of c-myc was ascertained by reincubating the blot with the same antibody used for IP. ( D , left panel). HuR binding to myosin IIA is sensitive toward RNase treatment. HMC overexpressing c-myc-HuRwild-type or c-myc-HuRΔHinge were treated for 2 h with AngII (0.1 µM) before total cell lysates from transfectants were immunoprecipitated with an anti-c-myc antibody or with control IgG. Before the IP reaction, the cell homogenates were subjected to RNase treatment and subsequently immunoblotted with an anti-myosin IIA specific antibody. Equal pull-down of c-myc is ascertained by reincubating the blot with anti-c-myc antiserum. (right panel). To test for AngII-dependent association of myosinIIA with endogenous HuR, 200 µg of total cell extracts from unstimulated (−) or AngII-stimulated (+) HMC were subjected to (IP) as described in ‘Materials and Methods’ section. The data shown are from a single experiment representative of two repeats with similar results.

    Article Snippet: Antibodies raised against HuR, histone deacetylase-1 (HDAC1), c-myc, anti-rabbit and anti-mouse horseradish peroxidase linked immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Overlay Assay, Binding Assay, Co-Immunoprecipitation Assay

    AngII promotes translocation of PKCδ to the nucleus and induces a physical interaction between PKCδ and nuclear HuR. (A) Indirect immunofluorescence images show PKCδ (green) and HuR (red) proteins in unstimulated cells and cells stimulated for 15 min with AngII (100 nM) as indicated. The merged images show a colocalization of PKCδ and HuR after AngII exposure (yellow signal). Data are representative of two independent experiments giving similar results. (B) Time course of AngII-induced PKCδ entry to the nucleus. Quiescent hMC were treated with either vehicle (−) or with AngII (100 nM) for the indicated time points and then lysed. Fifty micrograms of cytosolic (PKCδ c ) and nuclear (PKCδ n ) extracts was subjected to SDS-PAGE and successively immunoblotted with a phosphorylation-independent PKCδ antibody. To ascertain equal protein contents within the fractions, the blots were stripped and reprobed with an HDAC1-specific or anti-β-actin antibody. The Western blots shown are representative of three independent experiments giving similar results. (C) IP of nuclear PKCδ (PKCδ) and pSer-PKC substrate (pSer) levels in hMC stimulated either without (−) or with (+) 100 nM AngII for 15 min. For IP, a total protein amount (500 μg) of nuclear extracts from differentially treated cells was incubated overnight with 2 μg of either anti-HuR antibody (a.-HuR) or a nonspecific IgG isotype antibody (IgG). Equal amounts of immunoprecipitated HuR were ascertained by stripping the blot and reincubating it with the anti-HuR antibody used for IP. The data shown are representative of three independent experiments giving similar results.

    Journal: Molecular and Cellular Biology

    Article Title: Posttranslational Modification of the AU-Rich Element Binding Protein HuR by Protein Kinase Cδ Elicits Angiotensin II-Induced Stabilization and Nuclear Export of Cyclooxygenase 2 mRNA

    doi: 10.1128/MCB.01530-07

    Figure Lengend Snippet: AngII promotes translocation of PKCδ to the nucleus and induces a physical interaction between PKCδ and nuclear HuR. (A) Indirect immunofluorescence images show PKCδ (green) and HuR (red) proteins in unstimulated cells and cells stimulated for 15 min with AngII (100 nM) as indicated. The merged images show a colocalization of PKCδ and HuR after AngII exposure (yellow signal). Data are representative of two independent experiments giving similar results. (B) Time course of AngII-induced PKCδ entry to the nucleus. Quiescent hMC were treated with either vehicle (−) or with AngII (100 nM) for the indicated time points and then lysed. Fifty micrograms of cytosolic (PKCδ c ) and nuclear (PKCδ n ) extracts was subjected to SDS-PAGE and successively immunoblotted with a phosphorylation-independent PKCδ antibody. To ascertain equal protein contents within the fractions, the blots were stripped and reprobed with an HDAC1-specific or anti-β-actin antibody. The Western blots shown are representative of three independent experiments giving similar results. (C) IP of nuclear PKCδ (PKCδ) and pSer-PKC substrate (pSer) levels in hMC stimulated either without (−) or with (+) 100 nM AngII for 15 min. For IP, a total protein amount (500 μg) of nuclear extracts from differentially treated cells was incubated overnight with 2 μg of either anti-HuR antibody (a.-HuR) or a nonspecific IgG isotype antibody (IgG). Equal amounts of immunoprecipitated HuR were ascertained by stripping the blot and reincubating it with the anti-HuR antibody used for IP. The data shown are representative of three independent experiments giving similar results.

    Article Snippet: Antibodies raised against β-actin, COX-2, HuR, and histone deacetylase 1 (HDAC-1) and anti-rabbit and anti-mouse horseradish peroxidase-linked immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: Translocation Assay, Immunofluorescence, SDS Page, Western Blot, Incubation, Immunoprecipitation, Stripping Membranes

    AngII induces the RNA binding of HuR to the UTR of COX-2. hMC were stimulated with vehicle (−) or with 100 nM AngII (+) for 2 h before cells were cross-linked and lysed for IP. HuR-bound mRNA was precipitated by addition of a HuR-specific antibody or, alternatively, the same amount of IgG as a negative control. After elution from Sepharose beads, the isolated RNA samples were analyzed by RT-PCR either by using primer pairs, complementary to sequences encompassing different COX-2 3′ UTR parts (UTR1 and UTR2), or by use of a primer pair complementary to a sequence from the CR as described in Materials and Methods. Input GAPDH levels isolated before the IP were used as a control for equal amounts of input RNA (input) and were assessed by RT-PCR. Furthermore, input HuR levels isolated prior to the IP were used as input protein (input) control. A PCR without RT product was used as a negative control (no RT), whereas COX-2 full-length plasmid used as a template was used as a positive control (cDNA).

    Journal: Molecular and Cellular Biology

    Article Title: Posttranslational Modification of the AU-Rich Element Binding Protein HuR by Protein Kinase Cδ Elicits Angiotensin II-Induced Stabilization and Nuclear Export of Cyclooxygenase 2 mRNA

    doi: 10.1128/MCB.01530-07

    Figure Lengend Snippet: AngII induces the RNA binding of HuR to the UTR of COX-2. hMC were stimulated with vehicle (−) or with 100 nM AngII (+) for 2 h before cells were cross-linked and lysed for IP. HuR-bound mRNA was precipitated by addition of a HuR-specific antibody or, alternatively, the same amount of IgG as a negative control. After elution from Sepharose beads, the isolated RNA samples were analyzed by RT-PCR either by using primer pairs, complementary to sequences encompassing different COX-2 3′ UTR parts (UTR1 and UTR2), or by use of a primer pair complementary to a sequence from the CR as described in Materials and Methods. Input GAPDH levels isolated before the IP were used as a control for equal amounts of input RNA (input) and were assessed by RT-PCR. Furthermore, input HuR levels isolated prior to the IP were used as input protein (input) control. A PCR without RT product was used as a negative control (no RT), whereas COX-2 full-length plasmid used as a template was used as a positive control (cDNA).

    Article Snippet: Antibodies raised against β-actin, COX-2, HuR, and histone deacetylase 1 (HDAC-1) and anti-rabbit and anti-mouse horseradish peroxidase-linked immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: RNA Binding Assay, Negative Control, Isolation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction, Plasmid Preparation, Positive Control