Validation of Endothelin B Receptor Antibodies Reveals Two Distinct Receptor-related Bands on Western Blot.

Authors:
Barr TP, Kornberg D, Montmayeur JP, Long M, Reichheld S, Strichartz GR.
In:
Source: Anal Biochem
Publication Date: (2014)
Issue: ePub: ePub
Research Area:
Neurobiology
Basic Research
Cells used in publication:
293
Species: human
Tissue Origin: kidney
Astrocyte, rat
Species: rat
Tissue Origin: brain
Culture Media:
Experiment


Abstract

Antibodies are important tools for the study of protein expression, but are often used without full validation. In this study, we use Western blots to characterize antibodies targeted to the N- (NT) or C-termini (CT) and the second (IL2) or third intracellular (IL3) loops of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50kD band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37 kD band, but failed to detect endogenous ETB in rat brain. Bands detected by the CT-targeted or IL3-targeted antibodies were found to be unrelated to ETB. Our findings show that functional ETB receptors can be detected at 50 kD or 37 kD on Western blot, with drastic differences in antibody affinity for these bands. The 37 kD band likely reflects ETB receptor processing, which appears to be dependent on cell type and/or culture condition.