Fig 2: Activation of EMR2 by an antibody. NFAT-Luciferase reporter (NFAT-Luc) responses were measured following transient co-transfection of Vector, EMR2-FL, EMR2-CTF or AT-1 constructs with an NFAT-Luciferase reporter construct into HEK 293 T cells, and treatment with EMR2 antibodies (5–10 mg/ml) or Ang II (25 µM) as indicated: (panel a) pAb R&D Systems AF4894, (panel b) Serotec mAb, 2A1. Data were normalised as fold effect relative to vector control. (Panel c) illustrates the NFAT-Luciferase activity (RLU, Relative Light Units) in EMR2-FL transfected cells treated with pAb EMR2 (R&D Systems), at the indicated antibody concentrations.

Fig 3: Detection of EMR2 by commercial antibodies. HEK 293 T cells were transfected with a control BacMam virus (V) or with BacMam virus engineered to produce EMR2 (E). Protein size markers are indicated (M). Immunoprecipitation (IP) was carried out from cell lysates using R&D Systems anti-EMR2 pAb AF4894 (panel a), or Serotec mAb 2A1 (panel b) and immunoprecipitates were subjected to electrophoresis on the same gel. Following transfer, the membrane was divided into two strips which were probed with (a) 2A1, or (b) AF4894, respectively. These strips are presented in their entirety side by side as panels a and b. The specific EMR2 band is boxed in each case.

Fig 4: EMR2 and CD97 stimulate G protein coupling in a yeast-based reporter assay. Constructs expressing full-length (-FL) or truncated (-CTF) forms of EMR2 (a) and CD97 (b) were transformed into yeast strains expressing yeast Gpa1p or chimeric Gpa1/G protein alpha subunits corresponding to mammalian Ga sequences as designated on the x axis. Fluorescence Intensity Units (ex. 485 nm/em. 535 nm) report receptor-stimulated cell growth leading to the production of fluorescein. Data are presented as fold increase over a vector control. (c) Responses induced by EMR2-FL and EMR2-CTF were compared with human somatostatin receptor unstimulated (sst2) or stimulated (sst2 + SRIF) with a saturating concentration (1 µM) of somatostatin (SRIF-14). All data are plotted as the mean + range of four independent isolates, two replicates of each.

Fig 5: Activation of Inositol phosphate production by EMR2. EMR2-FL, EMR2-CTF and vector control constructs were transfected into HEK 293 T cells in combination with different G protein alpha subunits, as indicated, and tested in the IP-One HTRF assay (Cisbio). A reduction in the ratio (665 nm/615 nm) indicates an accumulation of inositol monophosphate, a downstream metabolite of IP3 induced by activation of a phospholipase C cascade. Data represent mean ± SD of 16 replicates for each condition. Statistical significance was measured using unpaired t-test (*p < 0.05).