Ptt5 vector

NMU2 constructs of WT and mutants were constructed into PTT5 vector (Invitrogen), as mentioned above. HEK 293F cells were transiently transfected with WT or mutants with total of 2000 ng plasmid DNA using transfection reagent (PEI MAX 2000, Polysciences) and cultivated at 37 °C with 5% CO₂. Cells were collected after 48-h transfection. IP accumulation was tested by IP-One Gq assay kit (Cisbio Bioassays, 62IPAPEJ) following the instruction manual. In general, cells were resuspended with 1× HBSS buffer containing 20 mM LiCl (stimulation buffer) and seeded into the white 384-well microplates (Proxi Plate^TM-384 Plus, PerkinElmer) with a density of 20,000 per well. Cells were preincubated with 10⁻⁵ M R-PSOP (provided by Boehringer Ingelheim company, Germany) and gradient concentration from 10⁻⁴ to 10⁻¹¹ M of NMU-25 or gradient concentration 10⁻⁴ to 10⁻¹¹ M of NMU-25 alone in stimulation buffer at 37 °C for 90 min. The cryptate-labeled anti-IP1 monoclonal antibody and d2-labeled IP1 were diluted with lysis & detection buffer (1:20) and added to each well by 3 μl, respectively. The plates were incubated at room temperature for 60 minutes and then read in a Synerg^TM H1 microplate reader (BioTek) with excitation at 330 nm and emission at 620 and 665 nm. The IP1 production was calculated by a standard dose–response curve and analyzed by GraphPad Prism 8.0.

The gene of human FPR2 was codon-optimized and synthesized by Genewiz. It was cloned into a modified pTT5 vector (Invitrogen) containing an expression cassette with a hemagglutinin (HA) signal sequence followed by a Flag tag at the N terminus and a PreScission protease site and a 10 × His-tag at the C terminus. The N-terminal residues M1-E2 of FPR2 were replaced by the fusion partner bRIL using overlap extension PCR. Five residues (E347–M351) were truncated at the C terminus. A point mutation S211^5.48L was introduced into the FPR2 gene by standard QuikChange PCR. The codon-optimized DNA sequence and all primer sequences are shown in Supplementary Table 4.
HEK293F cells (Invitrogen) were grown in suspension with the starting density at 0.6 × 10⁶ cells ml⁻¹ in 5% CO₂ at 37 °C. Once the cell density was increased to 1.2 × 10⁶ cells ml⁻¹, the cells were transfected with the plasmid of FPR2 using the FreeStyle^TM 293 Expression system (Invitrogen). Cells were collected after 48 h post transfection by centrifugation, and then stored at –80 °C until further use.