Figure 1.

Purification and validation of GPR39 as a putative receptor for 14,15-EET. A: chemoproteomics strategy to identify 14,15-EET receptor in mVSMCs. B: photo-crosslinking GPR39 with EET-P in mVSMCs. Left: representative SDS-PAGE gel revealing the presence of a ∼50 kDa band. 14,15-EET pretreatment reduced protein binding to EET-P in a dose-dependent manner. Right: total protein determined by Coomassie Blue staining of the same gel. C: representative confocal images demonstrating binding of EET-P to outer surface of mVSMCs (left, red). Pretreatment with 1 μM 14,15-EET for 10 min prevented EET-P surface binding (right; blue color is nuclear stain DAPI). D: a representative immunofluorescent confocal image illustrating GPR39 expression (left, red) in primary cultured mouse heart mVSMCs (scale bar = 20 μm), and expression of GPR39 1a, but not 1b, confirmed by qPCR (right), n = 3. E: a representative Western blot of EET-P cross linking to epitope-tagged GPR39 1a in transfected HEK cells. HEK cells were treated with 5 μM 14,15-EET or 14,15-EEZE for 10 min, 1 μM EET-P for 15 min, and then irradiated with UV for 5 min at 4oC. After clicking with biotin and purification with streptavidin Dynabeads, protein extracts were probed by anti-HA antibody. F: a representative dot-blot assay and quantification of dose-dependent binding of 14,15-EET, but not 11,12-EET, to membrane protein extracts from HEK-293 cells stably expressing GPR39 1a, but not untransfected control cells (top, raw image, and optical density quantification, bottom; n = 3). EET, epoxyeicosatrienoate; EET-P, epoxyeicosatrienoate probe; GPR39, G protein-coupled receptor 39; HEK, human embryonic kidney; mVSMCs, microvascular smooth muscle cells; 14,15-EEZE, 14,15-epoxyeicosa-5(Z)-enoic acid.