Figure 3
The choroid plexus expresses augurin, a 14 kDa processed protein form of the ECRG4 ORF corresponding to amino acids 31-148. A: Immunostaining in rat brain: Immunoreactive augurin was present in the choroid plexus (arrow) and ependyma (arrowhead) of the third ventricle in the adult rat compared to pre-immune IgY control (top inset). Scale bar = 20 μm B: Immunostaining in human brain: Adult human tissue showed that similarly, augurin staining localized primarily to the epithelial cell layer of the human CP (black arrow). Scale bar = 20 μmC: Immunoblotting of rat choroid plexus. When rat CP tissue was extracted and analyzed by immunoblotting as described in the text, an immunoreactive 14 kDa band was detected. It corresponds to the anticipated molecular weight of augurin. D: Augurin secretion in vitro: When adult primary human CPe cells were transduced with an adenovirus vector containing the green fluorescent protein gene (AdGFP), they were fluorescent (not shown) and like untreated CP cells in culture, do not express augurin. When these cells are transduced with adenovirus vector containing Ecrg4 cDNA (Ad Ecrg4 ), augurin protein can be detected in RIPA lysates by immunoblotting and the Ad ECRG4 -transduced CPe cells secrete augurin into the conditioned medium that can be measured by ELISA but not detected by immunoblotting. M = molecular weight markers and rAugurin = recombinant augurin used as standard and purified from, E coli.