Inositol phosphate accumulation assay

Flag-tagged wild-type and mutant FPR2s were cloned into the expression vector pTT5 (Invitrogen) and expressed in HEK293 cells (Invitrogen) along with the chimeric Gα protein Gα_Δ6qi4myr at the ratio of plasmids of 1:2 (w/w). Cells were routinely tested for mycoplasma contamination. Cells were harvested 48 h post transfection. Cell-surface expression of the receptor was measured by mixing 10 μl cells and 15 μl Monoclonal ANTI-FLAG M2-FITC antibody (Sigma, F4049; 1:100 diluted by TBS supplemented with 4% BSA). After 20 min, the fluorescence signal on the cell surface was measured by a FCM (flow cytometry) reader (Millipore).

IP1 accumulation was measured using an IP-One Gq assay kit (Cisbio Bioassays, 62IPAPEB) following the manufacturer’s instruction. In brief, the cells were plated in 384-well plates (20,000 cells per well) and treated with different concentrations of WKYMVm (1 pM–10 μM) or fMLFK (1 nM–1 mM) diluted in stimulation buffer at 37 °C for 90 min. Then 3 μl cryptate-labeled anti-IP1 monoclonal antibody and 3 μl d2-labeled IP1, which were pre-diluted in Lysis Buffer (1:20), were added to the wells, and incubated at room temperature for 1 h. Plates were read in a Synergy^TM H1 Operator (BioTek) with excitation at 330 nm and emission at 620 and 665 nm. The IP1 production was calculated according to a standard dose–response curve. Data were analyzed using Prism 7.0.