Adi 900 066

CSF levels of cAMP and cGMP were determined by ELISA. Both cAMP and cGMP ELISAs were obtained from Enzo Lifesciences (cAMP: #ADI-900-066; cGMP: #ADI-900-014) and used according to the manufacturer’s protocol. Briefly, for cAMP, 100 μl standard or CSF and 100 μl 0.1 M HCl were added into the appropriate wells. For cGMP, 200 μl CSF and 200 μl 0.1 M HCl were used. After adding 50 μl of the conjugate and 50 μl of the antibody to the appropriate wells, the plates were incubated at room temperature for 2 h with shaking. After washing steps, pNpp substrate and stop solution were added and the optical density was measured at 405 nm. The assays were blind for patient identification and disease status.

Mouse sperms at a density of 2 × 10⁷ cells/ml (6 × 10⁶ cells in total) were first peptide transduced with TAT-GIV-CT for 30 min, washed gently with PBS three times to remove excess peptides before their use in cAMP assays. Peptide-transduced sperms were pre-incubated with 0.5 mM isobutyl methyl xanthine (IBMX) prior to exposure to various chemicals at the following final concentrations: 25 mM NaHCO_3, 0.1 mM adenosine, or 100 µM. After mixing with the respective stimulus, the samples were incubated for 30 min at 37°C, followed by the addition of 0.25 M HCl (final concentration) to quench the biochemical reactions. After incubation for 30 min at room temperature, cell debris was sedimented by centrifugation at 3000 g for 5 min at room temperature. The cAMP concentration in the supernatant was determined by a competitive enzyme immunoassay according to the product manual (catalog # ADI-900-066, Enzo Life Sciences).

The effects of oxyntomodulin analogues and native GLP-1, glucagon and oxyntomodulin on cyclic adenosine monophosphate (cAMP) accumulation were assessed in Chinese hamster ovary (CHO-K1) cells over-expressing the rat glucagon receptor (cDNA from Origene, Maryland, USA) and Chinese hamster lung (CHL) cells over-expressing the rat GLP-1 receptor (a kind gift from Professor Bernard Thorens, University of Lausanne, Switzerland). For cAMP studies, cells were seeded at a density of 150000 cells/ml in a 48 well plate (Nunc, VWR International Inc., Chicago, USA) and incubated for 24 h. Cells were serum starved for 1 h and then incubated for 30 mins at room temperature with a range of peptide concentrations diluted in serum free DMEM (Sigma–Aldrich, Dorset, UK) in the presence of 1 mM IBMX (3-isobutyl-1-methylxanthine, Sigma–Aldrich). After incubation, medium was removed and cells lysed with 0.1 M HCl +0.5% Triton-X. cAMP levels were measured using an ELISA kit according to manufacturer’s instructions (ADI-900-066, Enzo Life Sciences, New York, USA), optical density was read at 405 nm on a Biotek ELx808 (Wolf Laboratories, York, UK). EC₅₀’s were calculated using Prism v5 (GraphPad Software Inc., San Diego, CA, USA). Concentrations were applied in duplicate or triplicate per experiment and each experiment repeated a minimum of 3 times.

Adherent HEK293-T cells were transiently transfected with either a plasmid encoding mouse Adgrd1, or a negative control encoding membrane-tethered EGFP (vector), or were treated with the transfection reagent Lipofectamine 2000 alone (mock). Twenty-four hours after transfection, cells were seeded on a streptavidin-coated microtitre plate that had been pre-incubated with the mouse PLXDC2 or control rat Cd200 biotinylated ectodomains. After three hours at 37 °C, the levels of cyclic AMP were determined in duplicate using a cAMP ELISA kit (ADI-900-066, Enzo Life Science) according to the manufacturer’s protocol, and normalised to the total protein concentration determined using the Bradford assay (ThermoFisher Scientific). One-way ANOVA showed a significant variation among conditions (F₅,₁₂ = 43, p < 0.0001) and a post-hoc Bonferroni test indicated that intracellular cAMP differed significantly in Adgrd1-transfected cells treated with Plxdc2 compared to Adgrd1-transfected cells treated with the control protein CD200 (p = 0.0391).