Cmpd101

PACAP (PACAP-38) was purchased from Peptide Institute (Minoh, Osaka, Japan). H89 and CMPD101 were purchased from Tocris Bioscience (Bristol, UK). D-sphingosine and Concanavarin A (ConA) were purchased from Sigma-Aldrich (St Louis, MO, USA) and Pitstop 2 (ab120687) was purchased from Abcam (Cambridge, UK).

CXCL12 was obtained from Almac. CMPD101 was obtained from Tocris Bioscience. Hanks’ balanced salt solution, Dulbecco’s Modified Eagle medium and penicillin/streptomycin were obtained from Gibco. Fetal bovine serum was obtained from Bodinco (Alkmaar, Netherlands). Coelenterazine-h was obtained from Promega (Madison, WI, USA).

Internalization assays were performed using the Nano-Glo HiBiT extracellular detection system acquired from Promega Corporation (Cat. No. N2421). The day before transfection, HEK293 cells were seeded in 96-well plates at a density of 2.5 × 10⁴ cells per well. The following day, cells were transfected with a mixture prepared containing 0.5 ng of SmBiT-high-affinity-Galr2 construct and 0.2 μl Lipofectamine 2000 (Invitrogen) added to each well. At 24 h post transfection, wells allocated for inhibitor pretreatment cells were pretreated with 30 μM of GRK2/3 inhibitor cmpd101 from Tocris Bioscience (Cat. No. 5642) for 30 min, 25 μM of clathrin inhibitor PitStop 2 from Abcam (Cat. No. ab120687) for 15 min, and 80 μM dynamin inhibitor Dynasore from Abcam (Cat. No. ab120192) for 40 min, and then treated using different agonist concentrations for 30 min, immediately after 100 μl of Nano-Glo HiBiT extracellular reagent (1 μl of LgBiT protein + 2 μl Substrate + 97 μl of Nano-Glo HiBiT buffer) was added and left to equilibrate for 4 min at room temperature without mixing. Then luminescence values were recorded immediately (Synergy 2 Multi-Mode Microplate Reader BioTek). Luminescent values were normalized in reference to cells treated with vehicle only.

Norbinaltorphimine (norBNI) and U50,488 (NIDA Drug Supply program) were dissolved in sterile saline (0.9%) at injected at 10 mL/kg (i.p.). The GRK2/3 inhibitor CMPD101 (Tocris Bioscience) was used as described (Abraham et al., 2018 (link)).