Pei max 2000

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.

HEK293F cells were transiently transfected with WT or mutant SSTR2/SSTR4 using transfection reagent (PEI MAX 2000, Polysciences) and incubated at 37 °C in 5% CO₂. Forty-eight hours after transfection, cells were centrifuged and resuspended in stimulation buffer (Hanks’ balanced salt solution (HBSS) supplemented with 5 mM HEPES, 0.5 mM IBMX and 0.1% (w/v) bovine serum albumin (BSA), pH 7.4) to a density of 200,000 cells/mL and added to 384-well white plates (PerkinElmer, 1000 cells/well). cAMP accumulation was measured by a LANCE Ultra cAMP kit (PerkinElmer) according to the manufacturer’s instructions. In brief, transfected cells were incubated for 40 min in stimulation buffer with 2.5 μL forskolin (SSTR2: 2 μM; SSTR4: 4 μM) and gradient concentration of ligand at RT. The reactions were stopped by addition of lysis buffer containing 5 μL Eu-cAMP tracer and 5 μL ULight-anti-cAMP. Plates were then incubated for 60 min at RT and time-resolved FRET signals were measured at 620 nm and 665 nm by an EnVision multilabel plate reader (PerkinElmer). Data were analyzed in GraphPad PRISM 8 and all values were normalized to the WT for each ligand. All outcomes were repeated at least three times.