at2  (Alomone Labs)


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    Alomone Labs at2
    Regulation of VEGF, fibronectin and laminin β1 expression in kidneys from hyperglycemic AT2KO mice A. <t>AT2</t> protein level was measured by immunoblot using a specific antibody. The lower panel shows combined data obtained on kidneys from 5 individual mice for each experimental condition. B. VEGF protein (B) and mRNA (C) levels were measured by immunoblot and RT-qPCR, respectively, on the same number of kidneys as in panel A. Identical experiments were performed for the protein and mRNA levels of fibronectin (D - E) and laminin β1 (F - G). **p
    At2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)"

    Article Title: Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2010.07.012

    Regulation of VEGF, fibronectin and laminin β1 expression in kidneys from hyperglycemic AT2KO mice A. AT2 protein level was measured by immunoblot using a specific antibody. The lower panel shows combined data obtained on kidneys from 5 individual mice for each experimental condition. B. VEGF protein (B) and mRNA (C) levels were measured by immunoblot and RT-qPCR, respectively, on the same number of kidneys as in panel A. Identical experiments were performed for the protein and mRNA levels of fibronectin (D - E) and laminin β1 (F - G). **p
    Figure Legend Snippet: Regulation of VEGF, fibronectin and laminin β1 expression in kidneys from hyperglycemic AT2KO mice A. AT2 protein level was measured by immunoblot using a specific antibody. The lower panel shows combined data obtained on kidneys from 5 individual mice for each experimental condition. B. VEGF protein (B) and mRNA (C) levels were measured by immunoblot and RT-qPCR, respectively, on the same number of kidneys as in panel A. Identical experiments were performed for the protein and mRNA levels of fibronectin (D - E) and laminin β1 (F - G). **p

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR

    VEGF mRNA translation in hyperglycemic kidneys A. VEGF mRNA translation was studied by polysome assay. The shift of VEGF mRNA toward the heaviest fractions in diabetic kidneys indicates active translation, and is reversed by the AT2 but not the AT1 antagonist. Note that the distribution of GAPDH mRNA is unaffected by the various treatments. The figure is representative of experiments performed on kidneys from 3 individual mice. B. Association of hnRNP K with VEGF mRNA was studied by immunoprecipitation with anti-hnRNP K antibody followed by extraction of associated RNA associated and RT-PCR. Note that hnRNP K is constitutively associated with both VEGF and GAPDH mRNAs and that only the association with VEGF mRNA is increased in diabetic kidneys. The figure is representative of experiments performed on kidneys from 3 individual mice. **p
    Figure Legend Snippet: VEGF mRNA translation in hyperglycemic kidneys A. VEGF mRNA translation was studied by polysome assay. The shift of VEGF mRNA toward the heaviest fractions in diabetic kidneys indicates active translation, and is reversed by the AT2 but not the AT1 antagonist. Note that the distribution of GAPDH mRNA is unaffected by the various treatments. The figure is representative of experiments performed on kidneys from 3 individual mice. B. Association of hnRNP K with VEGF mRNA was studied by immunoprecipitation with anti-hnRNP K antibody followed by extraction of associated RNA associated and RT-PCR. Note that hnRNP K is constitutively associated with both VEGF and GAPDH mRNAs and that only the association with VEGF mRNA is increased in diabetic kidneys. The figure is representative of experiments performed on kidneys from 3 individual mice. **p

    Techniques Used: Mouse Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Angiotensin receptors are expressed in normal murine kidneys AT1 (A) and AT2 (B) were detected in kidney slices as described in the methods section. Arrows indicate localization of AT1 and AT2. Magnification = 20X. AT1 is strongly expressed in the apical and lateral membranes of the tubules (thin arrows), and in mesangial cells (thick arrow),.AT2 is expressed mostly in glomerular endothelial cells (thick arrow) and in the proximal tubules (thin arrow). The pictures are representative of kidneys from 2 individual mice. C) detection of AT1 and AT2 by western blot in kidney cortex homogenates using the same antibodies as in A and B.
    Figure Legend Snippet: Angiotensin receptors are expressed in normal murine kidneys AT1 (A) and AT2 (B) were detected in kidney slices as described in the methods section. Arrows indicate localization of AT1 and AT2. Magnification = 20X. AT1 is strongly expressed in the apical and lateral membranes of the tubules (thin arrows), and in mesangial cells (thick arrow),.AT2 is expressed mostly in glomerular endothelial cells (thick arrow) and in the proximal tubules (thin arrow). The pictures are representative of kidneys from 2 individual mice. C) detection of AT1 and AT2 by western blot in kidney cortex homogenates using the same antibodies as in A and B.

    Techniques Used: Mouse Assay, Western Blot

    Signaling pathways activated by AT1 and AT2 in diabetic kidneys The diagram depicts a summary of the data obtained in this study. AT1 activation by hyperglycemia activates mTOR independently of Akt and leads to inactivation of eEF2K but is not sufficient to cause the dephosphorylation and activation of eEF2. Activation of AT2 stimulates the Akt-mTOR pathway that inactivates eEF2K and activates PP2A that dephosphorylates eEF2; AT2 also mediates hyperglycemia-induced increment in eEF1A. Activation of eEF2 and up-regulation of eEF1A stimulate elongation phase of mRNA translation, leading to increased translation of VEGF mRNA.
    Figure Legend Snippet: Signaling pathways activated by AT1 and AT2 in diabetic kidneys The diagram depicts a summary of the data obtained in this study. AT1 activation by hyperglycemia activates mTOR independently of Akt and leads to inactivation of eEF2K but is not sufficient to cause the dephosphorylation and activation of eEF2. Activation of AT2 stimulates the Akt-mTOR pathway that inactivates eEF2K and activates PP2A that dephosphorylates eEF2; AT2 also mediates hyperglycemia-induced increment in eEF1A. Activation of eEF2 and up-regulation of eEF1A stimulate elongation phase of mRNA translation, leading to increased translation of VEGF mRNA.

    Techniques Used: Activation Assay, De-Phosphorylation Assay

    2) Product Images from "Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)"

    Article Title: Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2010.07.012

    Regulation of VEGF, fibronectin and laminin β1 expression in kidneys from hyperglycemic AT2KO mice A. AT2 protein level was measured by immunoblot using a specific antibody. The lower panel shows combined data obtained on kidneys from 5 individual mice for each experimental condition. B. VEGF protein (B) and mRNA (C) levels were measured by immunoblot and RT-qPCR, respectively, on the same number of kidneys as in panel A. Identical experiments were performed for the protein and mRNA levels of fibronectin (D - E) and laminin β1 (F - G). **p
    Figure Legend Snippet: Regulation of VEGF, fibronectin and laminin β1 expression in kidneys from hyperglycemic AT2KO mice A. AT2 protein level was measured by immunoblot using a specific antibody. The lower panel shows combined data obtained on kidneys from 5 individual mice for each experimental condition. B. VEGF protein (B) and mRNA (C) levels were measured by immunoblot and RT-qPCR, respectively, on the same number of kidneys as in panel A. Identical experiments were performed for the protein and mRNA levels of fibronectin (D - E) and laminin β1 (F - G). **p

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR

    VEGF mRNA translation in hyperglycemic kidneys A. VEGF mRNA translation was studied by polysome assay. The shift of VEGF mRNA toward the heaviest fractions in diabetic kidneys indicates active translation, and is reversed by the AT2 but not the AT1 antagonist. Note that the distribution of GAPDH mRNA is unaffected by the various treatments. The figure is representative of experiments performed on kidneys from 3 individual mice. B. Association of hnRNP K with VEGF mRNA was studied by immunoprecipitation with anti-hnRNP K antibody followed by extraction of associated RNA associated and RT-PCR. Note that hnRNP K is constitutively associated with both VEGF and GAPDH mRNAs and that only the association with VEGF mRNA is increased in diabetic kidneys. The figure is representative of experiments performed on kidneys from 3 individual mice. **p
    Figure Legend Snippet: VEGF mRNA translation in hyperglycemic kidneys A. VEGF mRNA translation was studied by polysome assay. The shift of VEGF mRNA toward the heaviest fractions in diabetic kidneys indicates active translation, and is reversed by the AT2 but not the AT1 antagonist. Note that the distribution of GAPDH mRNA is unaffected by the various treatments. The figure is representative of experiments performed on kidneys from 3 individual mice. B. Association of hnRNP K with VEGF mRNA was studied by immunoprecipitation with anti-hnRNP K antibody followed by extraction of associated RNA associated and RT-PCR. Note that hnRNP K is constitutively associated with both VEGF and GAPDH mRNAs and that only the association with VEGF mRNA is increased in diabetic kidneys. The figure is representative of experiments performed on kidneys from 3 individual mice. **p

    Techniques Used: Mouse Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Angiotensin receptors are expressed in normal murine kidneys AT1 (A) and AT2 (B) were detected in kidney slices as described in the methods section. Arrows indicate localization of AT1 and AT2. Magnification = 20X. AT1 is strongly expressed in the apical and lateral membranes of the tubules (thin arrows), and in mesangial cells (thick arrow),.AT2 is expressed mostly in glomerular endothelial cells (thick arrow) and in the proximal tubules (thin arrow). The pictures are representative of kidneys from 2 individual mice. C) detection of AT1 and AT2 by western blot in kidney cortex homogenates using the same antibodies as in A and B.
    Figure Legend Snippet: Angiotensin receptors are expressed in normal murine kidneys AT1 (A) and AT2 (B) were detected in kidney slices as described in the methods section. Arrows indicate localization of AT1 and AT2. Magnification = 20X. AT1 is strongly expressed in the apical and lateral membranes of the tubules (thin arrows), and in mesangial cells (thick arrow),.AT2 is expressed mostly in glomerular endothelial cells (thick arrow) and in the proximal tubules (thin arrow). The pictures are representative of kidneys from 2 individual mice. C) detection of AT1 and AT2 by western blot in kidney cortex homogenates using the same antibodies as in A and B.

    Techniques Used: Mouse Assay, Western Blot

    Signaling pathways activated by AT1 and AT2 in diabetic kidneys The diagram depicts a summary of the data obtained in this study. AT1 activation by hyperglycemia activates mTOR independently of Akt and leads to inactivation of eEF2K but is not sufficient to cause the dephosphorylation and activation of eEF2. Activation of AT2 stimulates the Akt-mTOR pathway that inactivates eEF2K and activates PP2A that dephosphorylates eEF2; AT2 also mediates hyperglycemia-induced increment in eEF1A. Activation of eEF2 and up-regulation of eEF1A stimulate elongation phase of mRNA translation, leading to increased translation of VEGF mRNA.
    Figure Legend Snippet: Signaling pathways activated by AT1 and AT2 in diabetic kidneys The diagram depicts a summary of the data obtained in this study. AT1 activation by hyperglycemia activates mTOR independently of Akt and leads to inactivation of eEF2K but is not sufficient to cause the dephosphorylation and activation of eEF2. Activation of AT2 stimulates the Akt-mTOR pathway that inactivates eEF2K and activates PP2A that dephosphorylates eEF2; AT2 also mediates hyperglycemia-induced increment in eEF1A. Activation of eEF2 and up-regulation of eEF1A stimulate elongation phase of mRNA translation, leading to increased translation of VEGF mRNA.

    Techniques Used: Activation Assay, De-Phosphorylation Assay

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    Alomone Labs aar 012
    Angiotensin II AT 2 receptor immunocytochemistry in the mouse brain. (A) and (B) Antibody 2818-1 from Epitomics (dilution 1:400). The AT 2 receptor antibody reacts with parenchymal microvessels (indicated with arrowheads in A and B, and in box in panel A) in the cerebral motor cortex from -1.22 mm to Bregma, and the immunocytochemistry is similar in wild-type (WT) (A) and AT 2 knock-out (KO) mice (B). (C) and (D) Antibody sc-9040 from Santa Cruz (dilution 1:2000). The antibody detects ciliated ependymal cells (arrowheads) of the lateral ventricle (V), and the staining is similar in the wild type (C) or knockout mice (D) located at the same coordinates as A and B. (E) and (F): antibody <t>AAR-012</t> from Alomone (dilution 1:3000). In the cerebral motor cortex, the antibody detects cell colocalization with the neuronal marker NeuN (indicated with arrowheads in E and F and in the boxes in panel E). The staining is similar in wild-type (E) and AT 2 receptor knockout (F) mice. Scale bar = 20 µm.
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    Angiotensin II AT 2 receptor immunocytochemistry in the mouse brain. (A) and (B) Antibody 2818-1 from Epitomics (dilution 1:400). The AT 2 receptor antibody reacts with parenchymal microvessels (indicated with arrowheads in A and B, and in box in panel A) in the cerebral motor cortex from -1.22 mm to Bregma, and the immunocytochemistry is similar in wild-type (WT) (A) and AT 2 knock-out (KO) mice (B). (C) and (D) Antibody sc-9040 from Santa Cruz (dilution 1:2000). The antibody detects ciliated ependymal cells (arrowheads) of the lateral ventricle (V), and the staining is similar in the wild type (C) or knockout mice (D) located at the same coordinates as A and B. (E) and (F): antibody AAR-012 from Alomone (dilution 1:3000). In the cerebral motor cortex, the antibody detects cell colocalization with the neuronal marker NeuN (indicated with arrowheads in E and F and in the boxes in panel E). The staining is similar in wild-type (E) and AT 2 receptor knockout (F) mice. Scale bar = 20 µm.

    Journal: PLoS ONE

    Article Title: Commercially Available Angiotensin II At2 Receptor Antibodies Are Nonspecific

    doi: 10.1371/journal.pone.0069234

    Figure Lengend Snippet: Angiotensin II AT 2 receptor immunocytochemistry in the mouse brain. (A) and (B) Antibody 2818-1 from Epitomics (dilution 1:400). The AT 2 receptor antibody reacts with parenchymal microvessels (indicated with arrowheads in A and B, and in box in panel A) in the cerebral motor cortex from -1.22 mm to Bregma, and the immunocytochemistry is similar in wild-type (WT) (A) and AT 2 knock-out (KO) mice (B). (C) and (D) Antibody sc-9040 from Santa Cruz (dilution 1:2000). The antibody detects ciliated ependymal cells (arrowheads) of the lateral ventricle (V), and the staining is similar in the wild type (C) or knockout mice (D) located at the same coordinates as A and B. (E) and (F): antibody AAR-012 from Alomone (dilution 1:3000). In the cerebral motor cortex, the antibody detects cell colocalization with the neuronal marker NeuN (indicated with arrowheads in E and F and in the boxes in panel E). The staining is similar in wild-type (E) and AT 2 receptor knockout (F) mice. Scale bar = 20 µm.

    Article Snippet: The antibody dilutions were as follows: sc-9040, 1:500; AAR-012, 1:200; 2818-1, 1:1000, as recommended by the manufacturers.

    Techniques: Immunocytochemistry, Knock-Out, Mouse Assay, Staining, Marker

    Expression of the AT1 receptor ( A ), AT2 receptor ( B ), and NOS3 ( C ) in kidney vessels from male and female mice on a normal sodium diet (all n = 5).

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Sex differences in acute ANG II-mediated hemodynamic responses in mice

    doi: 10.1152/ajpregu.00638.2009

    Figure Lengend Snippet: Expression of the AT1 receptor ( A ), AT2 receptor ( B ), and NOS3 ( C ) in kidney vessels from male and female mice on a normal sodium diet (all n = 5).

    Article Snippet: All blots were incubated overnight with the following primary antibodies: anti-AT1 receptor (Alomone Labs, Jerusalem, Israel), anti-AT2 receptor antibody (Alomone Labs), and anti-endothelial nitric oxide synthase 3 (NOS3) (BD Biosciences, San Jose, CA).

    Techniques: Expressing, Mouse Assay

    Expression of the AT1 receptor ( A ), AT2 receptor ( B ), and NOS3 ( C ), in kidney vessels from male and female mice on a low-sodium diet (all n = 5).

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Sex differences in acute ANG II-mediated hemodynamic responses in mice

    doi: 10.1152/ajpregu.00638.2009

    Figure Lengend Snippet: Expression of the AT1 receptor ( A ), AT2 receptor ( B ), and NOS3 ( C ), in kidney vessels from male and female mice on a low-sodium diet (all n = 5).

    Article Snippet: All blots were incubated overnight with the following primary antibodies: anti-AT1 receptor (Alomone Labs, Jerusalem, Israel), anti-AT2 receptor antibody (Alomone Labs), and anti-endothelial nitric oxide synthase 3 (NOS3) (BD Biosciences, San Jose, CA).

    Techniques: Expressing, Mouse Assay

    Expression of the AT1 receptor ( A ), AT2 receptor ( B ), and NOS3 ( C ) in kidney vessels from male and female mice on a high-sodium diet (all n = 5).

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Sex differences in acute ANG II-mediated hemodynamic responses in mice

    doi: 10.1152/ajpregu.00638.2009

    Figure Lengend Snippet: Expression of the AT1 receptor ( A ), AT2 receptor ( B ), and NOS3 ( C ) in kidney vessels from male and female mice on a high-sodium diet (all n = 5).

    Article Snippet: All blots were incubated overnight with the following primary antibodies: anti-AT1 receptor (Alomone Labs, Jerusalem, Israel), anti-AT2 receptor antibody (Alomone Labs), and anti-endothelial nitric oxide synthase 3 (NOS3) (BD Biosciences, San Jose, CA).

    Techniques: Expressing, Mouse Assay

    Schematic diagram illustrating the mechanistic links of Ang II-upregulation of nNOS protein expression in cardiac myocytes. Ang II stimulates AT1R in cardiomyocyte plasma membrane and quickly internalized, leading to activation of cardiac oxidases such as NADPH oxidase. Intracellular ROS, in turn , activates Akt and phosphorylates endogenous eNOS to activate the enzyme. Subsequently, S-nitrosation of AT2R near cysteine 349 residue contributes to AT2R translocation to the plasma membrane and was stimulated by Ang II. This leads to a rise in nNOS protein and NO generation

    Journal: Basic research in cardiology

    Article Title: ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes

    doi: 10.1007/s00395-015-0477-6

    Figure Lengend Snippet: Schematic diagram illustrating the mechanistic links of Ang II-upregulation of nNOS protein expression in cardiac myocytes. Ang II stimulates AT1R in cardiomyocyte plasma membrane and quickly internalized, leading to activation of cardiac oxidases such as NADPH oxidase. Intracellular ROS, in turn , activates Akt and phosphorylates endogenous eNOS to activate the enzyme. Subsequently, S-nitrosation of AT2R near cysteine 349 residue contributes to AT2R translocation to the plasma membrane and was stimulated by Ang II. This leads to a rise in nNOS protein and NO generation

    Article Snippet: Fixed cells (not permeabilized) were incubated in blocking solution (5 % FBS in PBS) for 1 h, followed by an incubation with extracellular membrane targeting anti-rabbit AT2R polyclonal antibody (1:100, Alomone labs) for overnight at 4 °C.

    Techniques: Expressing, Activation Assay, Translocation Assay

    S-nitrosation of AT2R is necessary for Ang II-stimulation of nNOS protein expression. a Representative immunoblots and mean ratios of AT2R S-nitrosation (relative to total AT2R) in the presence and absence of SNP (10 μM). S-nitrosated AT2R was increased by SNP. b DTT pre-treatment (1 mM, 30 min) abolished Ang II-induction of nNOS protein expression

    Journal: Basic research in cardiology

    Article Title: ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes

    doi: 10.1007/s00395-015-0477-6

    Figure Lengend Snippet: S-nitrosation of AT2R is necessary for Ang II-stimulation of nNOS protein expression. a Representative immunoblots and mean ratios of AT2R S-nitrosation (relative to total AT2R) in the presence and absence of SNP (10 μM). S-nitrosated AT2R was increased by SNP. b DTT pre-treatment (1 mM, 30 min) abolished Ang II-induction of nNOS protein expression

    Article Snippet: Fixed cells (not permeabilized) were incubated in blocking solution (5 % FBS in PBS) for 1 h, followed by an incubation with extracellular membrane targeting anti-rabbit AT2R polyclonal antibody (1:100, Alomone labs) for overnight at 4 °C.

    Techniques: Expressing, Western Blot

    AT2R antagonist blocks Ang II-stimulation of nNOS protein expression and activity in LV myocytes. a Isolated LV myocytes were pre-incubated with AT2R antagonist, PD123319 (1 μM). PD123319 blocked Ang II-stimulation of nNOS protein expression. b PD123319 prevented Ang II-induced increase in NO production. c AT2R agonist, CGP42112A (1 μM, 3 h), significantly increased nNOS protein expression

    Journal: Basic research in cardiology

    Article Title: ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes

    doi: 10.1007/s00395-015-0477-6

    Figure Lengend Snippet: AT2R antagonist blocks Ang II-stimulation of nNOS protein expression and activity in LV myocytes. a Isolated LV myocytes were pre-incubated with AT2R antagonist, PD123319 (1 μM). PD123319 blocked Ang II-stimulation of nNOS protein expression. b PD123319 prevented Ang II-induced increase in NO production. c AT2R agonist, CGP42112A (1 μM, 3 h), significantly increased nNOS protein expression

    Article Snippet: Fixed cells (not permeabilized) were incubated in blocking solution (5 % FBS in PBS) for 1 h, followed by an incubation with extracellular membrane targeting anti-rabbit AT2R polyclonal antibody (1:100, Alomone labs) for overnight at 4 °C.

    Techniques: Expressing, Activity Assay, Isolation, Incubation

    Time-dependent expressions of AT1R and AT2R in the surface membrane of LV myocytes following Ang II treatment (3 h) and the effect of ROS scavengers on their expression. a Representative AT1R and AT2R blottings before and after Ang II treatment (5, 10, 20 and 30 min) in LV myocytes. GAPDH was used as a loading control. b Immunoblottings and the mean ratios of AT1R and AT2R (surface/total) at 0, 10, 20 and 30 min after Ang II treatment. c Immunoblotting ( left ) and the mean ratio of AT2R (surface/total) ( right ). Losartan (1 μM), apocynin (100 μM) and tiron (1 mM) were pre-treated in LV myocytes, respectively

    Journal: Basic research in cardiology

    Article Title: ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes

    doi: 10.1007/s00395-015-0477-6

    Figure Lengend Snippet: Time-dependent expressions of AT1R and AT2R in the surface membrane of LV myocytes following Ang II treatment (3 h) and the effect of ROS scavengers on their expression. a Representative AT1R and AT2R blottings before and after Ang II treatment (5, 10, 20 and 30 min) in LV myocytes. GAPDH was used as a loading control. b Immunoblottings and the mean ratios of AT1R and AT2R (surface/total) at 0, 10, 20 and 30 min after Ang II treatment. c Immunoblotting ( left ) and the mean ratio of AT2R (surface/total) ( right ). Losartan (1 μM), apocynin (100 μM) and tiron (1 mM) were pre-treated in LV myocytes, respectively

    Article Snippet: Fixed cells (not permeabilized) were incubated in blocking solution (5 % FBS in PBS) for 1 h, followed by an incubation with extracellular membrane targeting anti-rabbit AT2R polyclonal antibody (1:100, Alomone labs) for overnight at 4 °C.

    Techniques: Expressing

    Cystein 349 is an important region for the translocation of AT2R to the plasma membrane. a Amino acid sequence and the mutated cysteine residues in AT2R. The AT2R protein has four cysteine residues in intracellular region. The arrowhead indicates the position where GFP was inserted to make GFP-tagged AT2R (AT2R-GFP). b HEK293T cells were transfected with AT2R-WT, C70A, C71A, C319A, C349A mutants, respectively. Each mutant was treated with SNP (10 μM). Quantification of the mutants were identified by GFP protein. Na + -K + ATPase (NKA) was detected as a loading control for surface fraction. Bar graph represents the ratio of surface to total GFP protein. c Overexpressed AT2R-GFP (green) was localized in the cytoplasm in the HEK293T cell. The surface expression of AT2R-GFP was identified by WGA ( red ). The right panel showed the intensities of AT2R-GFP ( green ) and WGA ( red ) on yellowed dashed lines of the images. Scale bar 5 μm

    Journal: Basic research in cardiology

    Article Title: ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes

    doi: 10.1007/s00395-015-0477-6

    Figure Lengend Snippet: Cystein 349 is an important region for the translocation of AT2R to the plasma membrane. a Amino acid sequence and the mutated cysteine residues in AT2R. The AT2R protein has four cysteine residues in intracellular region. The arrowhead indicates the position where GFP was inserted to make GFP-tagged AT2R (AT2R-GFP). b HEK293T cells were transfected with AT2R-WT, C70A, C71A, C319A, C349A mutants, respectively. Each mutant was treated with SNP (10 μM). Quantification of the mutants were identified by GFP protein. Na + -K + ATPase (NKA) was detected as a loading control for surface fraction. Bar graph represents the ratio of surface to total GFP protein. c Overexpressed AT2R-GFP (green) was localized in the cytoplasm in the HEK293T cell. The surface expression of AT2R-GFP was identified by WGA ( red ). The right panel showed the intensities of AT2R-GFP ( green ) and WGA ( red ) on yellowed dashed lines of the images. Scale bar 5 μm

    Article Snippet: Fixed cells (not permeabilized) were incubated in blocking solution (5 % FBS in PBS) for 1 h, followed by an incubation with extracellular membrane targeting anti-rabbit AT2R polyclonal antibody (1:100, Alomone labs) for overnight at 4 °C.

    Techniques: Translocation Assay, Sequencing, Transfection, Mutagenesis, Expressing, Whole Genome Amplification

    Effect of eNOS inhibition or gene deletion on AT2R translocation to plasma membrane and Ang II-stimulation of nNOS protein expression. a Ang II and SNP-dependent AT2R translocation to plasma membrane was detected by immunocytochemistry with anti-AT2R extracellular antibody ( green ) in non-permeabilized LV myocytes. Ang II and SNP increased AT2R expression in the plasma membrane, whereas L-NAME abolished AT2R translocation. b Surface biotinylation of AT2R after LV myocytes was treated by Ang II and Ang II + L-NAME. L-NAME prevented AT2R expression in plasma membrane. c L-NAME pre-treatment blocked Ang II-stimulation of nNOS protein expression. d Isolated LV myocytes in eNOS +/+ and eNOS −/− mice were treated with Ang II (1 μM) for 3 h. nNOS protein expression was increased by Ang II in eNOS +/+ but not in eNOS −/− LV myocytes

    Journal: Basic research in cardiology

    Article Title: ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes

    doi: 10.1007/s00395-015-0477-6

    Figure Lengend Snippet: Effect of eNOS inhibition or gene deletion on AT2R translocation to plasma membrane and Ang II-stimulation of nNOS protein expression. a Ang II and SNP-dependent AT2R translocation to plasma membrane was detected by immunocytochemistry with anti-AT2R extracellular antibody ( green ) in non-permeabilized LV myocytes. Ang II and SNP increased AT2R expression in the plasma membrane, whereas L-NAME abolished AT2R translocation. b Surface biotinylation of AT2R after LV myocytes was treated by Ang II and Ang II + L-NAME. L-NAME prevented AT2R expression in plasma membrane. c L-NAME pre-treatment blocked Ang II-stimulation of nNOS protein expression. d Isolated LV myocytes in eNOS +/+ and eNOS −/− mice were treated with Ang II (1 μM) for 3 h. nNOS protein expression was increased by Ang II in eNOS +/+ but not in eNOS −/− LV myocytes

    Article Snippet: Fixed cells (not permeabilized) were incubated in blocking solution (5 % FBS in PBS) for 1 h, followed by an incubation with extracellular membrane targeting anti-rabbit AT2R polyclonal antibody (1:100, Alomone labs) for overnight at 4 °C.

    Techniques: Inhibition, Translocation Assay, Expressing, Immunocytochemistry, Isolation, Mouse Assay