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Diabetologia (2014) 57:776780 DOI 10.1007/s00125-014-3161-8
SHORT COMMUNICATION
Detection of free fatty acid receptor 1 expression: the critical role of negative and positive controls
Charlott-Amelie Teutsch & Madhura Panse &
Manuel Grundmann & Gabriele Kaiser & Evi Kostenis &
Hans-Ulrich Hring & Susanne Ullrich
Received: 14 October 2013 /Accepted: 18 December 2013 /Published online: 26 January 2014 # Springer-Verlag Berlin Heidelberg 2014
AbstractAims/hypothesis Adequate evaluation of protein expression is a crucial prerequisite for functional studies. Commonly used strategies comprise detection of proteins by specific antibodies using western blotting and immunohistochemical staining, or detection of mRNA by in situ hybridisation and RT-PCR. We evaluated the tools for the detection of free fatty acid receptor 1 (FFAR1) expression.
Methods Commercially available antibody preparations were used to detect endogenous expression of the FFAR1 receptor and this was compared with cell preparations deficient or overexpressing the mouse or human receptor. Concentrations of mRNA were evaluated by RT-PCR.
Results All insulin-secreting cells, INS-1E, Min6 and mouse islets showed specific expression of Ffar1 at the mRNA level. However, none of the commercially available antibodies specifically detected rat, mouse or human FFAR1.
Conclusions/interpretation Proper positive and negative controls are an important prerequisite for the evaluation of FFAR1 expression.
Keywords Antibodies . FFAR1/GPR40 . Ffar1expression . RT-PCR . Western blotting
AbbreviationsDMR Dynamic mass redistributionFFAR1 Free fatty acid receptor 1GAPDH Glyceraldehyde-3-phosphate dehydrogenase HEK Human embryonic kidney 293HRP Horseradish peroxidaseIGF1R IGF-1 receptorPKB Protein kinase BTBS TRIS-buffered salineTUG-488 3-(4-((2-(Cyanomethyl)phenyl)ethynyl) phenyl)propanoic acidYFP Yellow fluorescent protein
Introduction
Free fatty acid receptor 1 (FFAR1; also known as G-protein-coupled receptor 40), encoded by Ffar1, is differentially expressed in organs, with a particularly high level of expression in the islets of Langerhans, where the receptor plays a stimula-tory role in glucose-induced insulin secretion [1]. To understand the role and function of FFAR1, especially during chronic exposure of beta cells to fatty acids, as occurs in obesity, detection of functional receptors at the plasma membrane is an important requirement. Repetitive or continuous stimulation has been found to result in receptor downregulation and internalisation which may impair receptor function [2, 3]. Indeed, receptor desensitisation to agonists after initial
Electronic supplementary material The online version of this article (doi:http://dx.doi.org/10.1007/s00125-014-3161-8
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