Coelentrazine h

HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.292 g/l L-glutamine and 10% (v/v) newborn-calf serum at 37°C in a 5% CO₂ humidified atmosphere. Cells were transfected with FLAG-tagged hGPR35 enhanced yellow fluorescent protein (FLAG-hGPR35-eYFP) and β-arrestin-2-Renilla luciferase 6 (ratio 4:1), using 1 mg/ml PEI. After 24 h, cells were washed with Hanks' balanced salt solution (pH 7.4), and coelentrazine-h (Promega) was added to a final concentration of 5 μM. Cells were incubated in darkness for 10 min at 37°C before the addition of receptor ligands. Cells were incubated for a further 5 min at 37°C before BRET measurements were performed using a PHERAstar FS reader (BMG-Labtech, Offenburg, Germany). The BRET ratio was calculated as a wavelength emission at 530/485 nm and expressed as the percentage of maximal signal for each ligand [13 (link),14 (link)].

Recombinant hCG and LH were purchased from National Peptides and Hormones Program (c/o A. F. Parlow, Harbor-UCLA Medical Center). For PALM studies, CAGE 500 and 552 N-hydroxysuccinimide esters for antibody conjugation and direct labeling of receptors were purchased from Abberior. Primary antibodies HA.11 and FLAG were purchased from Covance and Sigma, respectively. For BRET and pRL-cmv luciferase reporter assays, coelentrazine h and coelentrazine, respectively, were purchased from Promega. For cre-luc reporter gene assays, SteadyLite was purchased from PerkinElmer Life Sciences. Fluo-4 direct for calcium imaging was obtained from Invitrogen and HTRF-IP₁ assay from CisBio.

Luciferase assay was performed essentially as explained previously (Srikantha et al., 1996 (link)). Briefly, cells were harvested and extracted using RLUC buffer (0.5 M NaCl, 0.1 M K₂HPO₄ (pH), 1 mM Na2EDTA, 0.6 mM sodium azide, 1 mM phenylmethylsulfonyl fluoride, 0.02% bovine serum albumin). The assay was initiated by addition of 1.25 μM coelentrazine h (Promega) to cell extracts, and activity was measured using GloMax 20/20 luminometer (Promega). Luciferase activity (RLU) is expressed as relative luminescence per 10 s/mg protein. As a control, Luciferase mRNA was evaluated relative to GAPDH mRNA by qRT-PCR and no significant changes were elicited by any of the treatments used (data not shown).