Abstract
Several previous reports suggested that many commercially available antibodies directed against G protein-coupled receptors (GPCR) lack sufficient selectivity. Accordingly, it has been proposed that receptor antibodies should be validated by at least one of several criteria, such as testing tissues or cells after knockout or silencing of the corresponding gene. Here, we tested whether 12 commercially available antibodies directed against α-adrenergic receptor (AR) subtypes (α1A/B/D, α2A/B/C), atypical chemokine receptor 3 (ACKR3), and vasopressin receptor 1A (AVPR1A) suffice these criteria. We detected in flow cytometry experiments with human vascular smooth muscle cells that the fluorescence signals from each of these antibodies were reduced by 46 ± 10 %–91 ± 2 % in cells treated with commercially available small interfering RNA (siRNA) specific for each receptor, as compared with cells that were incubated with non-targeting siRNA. The tested antibodies included anti-ACKR3 (R&D Systems, mab42273), for which specificity has previously been demonstrated. Staining with this antibody resulted in 72 ± 5 % reduction of the fluorescence signal after ACKR3 siRNA treatment. Furthermore, staining with anti-α1A-AR (Santa Cruz, sc1477) and anti-ACKR3 (Abcam, ab38089), which have previously been reported to be non-specific, resulted in 70 ± 19 % and 80 ± 4 % loss of the fluorescence signal after α1A-AR and ACKR3 siRNA treatment, respectively. Our findings demonstrate that the tested antibodies show reasonable selectivity for their receptor target under our experimental conditions. Furthermore, our observations suggest that the selectivity of GPCR antibodies depends on the method for which the antibody is employed, the species from which cells/tissues are obtained, and on the type of specimens (cell, tissue/cell homogenate, or section) tested.
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This research was supported by the National Institute of General Medical Sciences (Awards R01GM107495 and T32GM008750); by the National Cancer Institute (Awards R01CA135341 and R01CA188427); by the National Heart, Lung, and Blood Institute (Award R21HL118588); and by a grant that was awarded and administered by the U.S. Army Medical Research and Materiel Command (USAMRMC)/Medical Research Acquisition Activity (USAMRAA) at Fort Detrick, MD, under Contract Number W81XWH-15-1-0262. The content is solely the responsibility of the authors.
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Tripathi, A., Gaponenko, V. & Majetschak, M. Commercially available antibodies directed against α-adrenergic receptor subtypes and other G protein-coupled receptors with acceptable selectivity in flow cytometry experiments. Naunyn-Schmiedeberg's Arch Pharmacol 389, 243–248 (2016). https://doi.org/10.1007/s00210-015-1196-0
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DOI: https://doi.org/10.1007/s00210-015-1196-0