at2  (Alomone Labs)

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

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

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

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

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

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

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

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

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

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