Ip one gq kit

Agonist-dependent inositol monophosphate (IP1) accumulation was measured using the IP-One G_q kit (Cisbio), a cell-based homogeneous time-resolved fluorescence assay, according to the manufacturer’s instructions. Briefly, cryo-preserved CHO cells stably expressing human OX₁R and human OX₂R were thawed, and resuspended in IMDM medium (Gibco) at densities of 2.0 × 10⁵ cells/ml and 4.0 ×10⁵ cells/ml, respectively. Cells were seeded into 384-well plates (50 µl per well) and incubated at 37 °C in 5% CO₂ for 20–24 h. In a separate plate, a dilution series of each agonist was prepared in Stimulation Buffer (Cisbio). The culture medium was removed and cells were incubated with 14 µl of agonist-containing Stimulation Buffer at 37 °C. After 1–2 h, 6 µl of Lysis and Detection Buffer (Cisbio) containing IP1-d2 and IP1 Tb cryptate antibody conjugates were added, and the reaction was incubated at room temperature protected from light. After 1 h, fluorescence was measured on an EnVision fluorescence plate reader (PerkinElmer) with the excitation wavelength set to 320 nm, and emission monitored at 620 nm (donor) and 665 nm (acceptor). Data were analyzed using GraphPad Prism.

cAMP: The test was conducted according to a cAMP Gs Dynamic Kit (CisBio, Paris, France). The fluorescence was measured (TECAN Infinitive M200 Pro plate reader (TECAN, Männedorf, Switzerland) with excitation at 340 nm and emission at 620 and 665 nm for two concentrations of the scFv_D2–5-HT1A protein: 33 μg/mL and 16.5 μg/mL for the four cell lines: HEK 293 and HEK 293 expressing either the D₂ or the 5-HT_1A receptor, or both of them.
IP₁: The test was conducted under the same conditions as the cAMP test, using an IP-One Gq Kit (CisBio).

For measuring IP accumulation, HEK293 cells grown to 70–80% confluency in 6-well plates were transiently co-transfected using Metafectene Pro (2.5 μl/μg DNA; Biontex) with plasmids containing receptor and/or mock (empty pcDNA3.1) (for endogenous G_q-coupling) using 4 μg total DNA in a ratio of 3:1 following the manufacturer’s instructions. For testing Gα₁₆- and Gα_Δ6qi4myr-coupling, plasmids encoding the corresponding Gα protein were co-transfected instead of mock. Gα₁₆ is naturally promiscuous and stimulates the phospholipase C (PLC) pathway leading to IP production, while the exchange of the C-terminal four amino acids of Gα_q for the corresponding residues of Gα_i1 in the chimeric Gα_Δ6qi4myr confers the ability to couple to G_i/o protein-preferring receptors but still stimulate PLC, thus specifically redirecting downstream cellular signaling [21 (link)]. 16 h post transfection, the cells were seeded into white 384-well plates at a density of 20,000 cells/well. Media was removed 24 h later and the cells were incubated with the indicated concentration of peptides for 1 h in HBSS + 20 mM LiCl. IP accumulation was measured with the HTRF-based IP-One Gq kit (Cisbio) on a microplate reader (Tecan Spark).

HEK 293 cells were seeded at 400,000 cells/well in 6-well plates. The next day, cells were transfected with 1 μg of GnRHR-WT or GnRHR-Ctail expression vectors using PEI transfection reagent in a mass ratio of 3:1 PEI to DNA. Twenty-four-hour post-transfection, cells were detached by manual pipetting and replated in 384-well low volume white plates (15,000 cells/well) and incubated for an additional 24 hr. Next, cells were washed and stimulated with 0, 10, or 100 nM GnRH for 30 min at 37°C. IP1 production was assessed using IP-ONE-Gq Kit (Cisbio, Codolet, France) following the manufacturer’s instructions. Homogeneous Time-Resolved Fluorescence (HTRF) was measured using a Synergy 2 Multi-Mode Microplate Reader (BioTek) and the ratio was calculated following the manufacturer’s instructions. Data are presented as relative HTRF, where values of stimulated conditions were normalized over the value of untreated GnRHR-WT expressing cells.

Expi293F cells stably expressing a tetracycline inducible wild-type human FLAG-AT1R were diluted to 1.5–2 × 10⁶ cells/mL and induced with 0.4 μg/mL doxycycline hyclate for 24–28 hours. 2 × 10⁴ cells were plated into a low-volume 96-well plate, treated with 5 μM of each AT118i4h32 variant for 30 min at 37 °C, and stimulated with AngII for 1 hour at 37 °C. IP1 was detected with the IP-One Gq kit (CisBio) and read on a SpectraMax M5e plate reader (Molecular Devices).