Lack of Specificity of Commercial Antibodies Leads to Misidentification of Angiotensin Type 1 Receptor ProteinNovelty and Significance by Marcela Herrera,

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Lack of Specificity of Commercial Antibodies Leads to Misidentification of Angiotensin Type 1 Receptor ProteinNovelty and Significance by Marcela Herrera, Matthew A. Sparks, Adolfo R. Alfonso-Pecchio, Lisa M. Harrison-Bernard, and Thomas M. Coffman Hypertension Volume 61(1):253-258 December 12, 2012 Copyright © American Heart Association, Inc. All rights reserved.

Western blot analysis of 40 μg homogenates of wild-type kidney cortex (C) and medulla (M) using 3 different commercially available angiotensin type 1 receptor (AT1R) antibodies. Western blot analysis of 40 μg homogenates of wild-type kidney cortex (C) and medulla (M) using 3 different commercially available angiotensin type 1 receptor (AT1R) antibodies. Antibody 1: Alomone AAR-011; antibody 2: Santa Cruz sc-1173 (n-10); antibody 3: Abcam 18801. X-ray films were exposed to the membranes for 2 min. Representative of n=2. Marcela Herrera et al. Hypertension. 2013;61:253-258 Copyright © American Heart Association, Inc. All rights reserved.

Western blot analysis of kidney cortex and medulla from wild-type mice (WT), AT1AKO mice (A), and AT1ABKO mice (AB) using 3 different commercially available anti-AT1R antibodies. Western blot analysis of kidney cortex and medulla from wild-type mice (WT), AT1AKO mice (A), and AT1ABKO mice (AB) using 3 different commercially available anti-AT1R antibodies. A, Antibody 1: Alomone AAR-011; B, antibody 2: Santa Cruz sc-1173 (n-10); C, antibody 3: AbCam 18801. Representative of n=3. Marcela Herrera et al. Hypertension. 2013;61:253-258 Copyright © American Heart Association, Inc. All rights reserved.

AT1R immunohistochemical localization using antibody 2 Santa Cruz N-10 rabbit polyclonal antibody (1:300) in kidney and liver of wild-type (WT) (A–D) mice and mice with combined deficiency of AT1A and AT1B receptors (AT1ABKO; E–H). AT1R immunohistochemical localization using antibody 2 Santa Cruz N-10 rabbit polyclonal antibody (1:300) in kidney and liver of wild-type (WT) (A–D) mice and mice with combined deficiency of AT1A and AT1B receptors (AT1ABKO; E–H). Consecutive tissue sections from WT kidney in the absence (A) of primary antibody demonstrate minimal background immunostaining compared with incubation of the tissue section in the presence of the AT1R (B). AT1R was localized to vascular smooth muscle cells (arrow) of WT and AT1ABKO mice in renal cortical (A–G) and liver (H) tissues. Proximal tubule brush border and basolateral membrane (double arrowhead) and distal nephron segments (asterisk) demonstrated positive AT1R immunostaining in WT and AT1ABKO mice. Images were obtained using a ×100 oil immersion lens. Bar, 50 μm. Marcela Herrera et al. Hypertension. 2013;61:253-258 Copyright © American Heart Association, Inc. All rights reserved.

A, CT values for AT1A receptor relative to GAPDH obtained by real-time polymerase chain reaction in kidney tissues from wild-type (WT), AT1AKO, and AT1ABKO mice. A, CT values for AT1A receptor relative to GAPDH obtained by real-time polymerase chain reaction in kidney tissues from wild-type (WT), AT1AKO, and AT1ABKO mice. B, Representative agarose gel to visualize AT1B receptor mRNA in WT, AT1AKO, and AT1ABKO. C, Effect of acute angiotensin II infusion (10 μg/kg) on blood pressure in WT, AT1AKO, and AT1ABKO mice. P<0.005 vs WT; n=3 to 4. Marcela Herrera et al. Hypertension. 2013;61:253-258 Copyright © American Heart Association, Inc. All rights reserved.

A, Cellular localization of AT1A in HEK cells cotransfected with expression plasmids encoding AT1A receptor fused with fluorescent mCherry (AT1A, magenta, I, III, IV, and VI) and with different plasmids encoding fluorescent markers for endoplasmic reticulum (Calreticulin, green, II and III) or Golgi apparatus (GalNAc, green, V and VI). A, Cellular localization of AT1A in HEK cells cotransfected with expression plasmids encoding AT1A receptor fused with fluorescent mCherry (AT1A, magenta, I, III, IV, and VI) and with different plasmids encoding fluorescent markers for endoplasmic reticulum (Calreticulin, green, II and III) or Golgi apparatus (GalNAc, green, V and VI). The fluorescent constructs were visualized after 24 h by live-cell confocal microscopy. AT1A receptors localize in the plasma membrane and partially associate with the Golgi apparatus. The results are representative of ≥2 independent experiments. B, Detection of AT1 receptors in HEK cells transfected with varying amounts (0, 2, 4, and 8 μg) of a plasmid encoding the His-tagged AT1A receptor (AT1A-His DNA) using different antibodies. Anti-His penta His antibody, Qiagen 34660; anti-AT1 receptor 1: Alomone AAR-011; anti-AT1 receptor 2: Santa Cruz sc-1173 (n-10); anti-AT1 receptor 3: AbCam 18801. Red boxes indicate bands corresponding to the AT1AR protein. Asterisk indicates a band recognized by the His antibody in nontransfected cells, suggesting an endogenous non-specific band. Marcela Herrera et al. Hypertension. 2013;61:253-258 Copyright © American Heart Association, Inc. All rights reserved.