Bglii restriction enzyme

DMEM (Dulbecco’s Modified Eagle Medium), FBS (fetal bovine serum), Collagenase Type I were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Restriction enzyme BglII and HindIII were bought from New England Biolabs (Ipswich, MA, USA). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA). Antibodies for C/EBPα, PPARγ and FABP4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for HSL, p-HSL, ERK, p-ERK, S6 and p-S6 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Methylisobutylxanthine, dexamethasone, Insulin, Oil Red O, Quinine hydrochloride dehydrate, Caffeine, Salicin, 6-propyl-2-thiouracil (PROP), Sucrose octaacetate, Hesperidin and Sodium Benzoate were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). All tested bitter compounds were selected based on common knowledge and previous publications mentioned above. The bitter agonists were either dissolved in DPBS or DMSO, and final DMSO concentration did not exceed 0.1% (v/v) to avoid toxic effect on cells.

HEK 293 cells (DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; RRID:CVCL_0045) were maintained in DMEM supplemented with FBS (Biochrom; Berlin Germany), penicillin (100 U·ml⁻¹; Biochrom), and streptomycin (100 μg·ml⁻¹; Biochrom), with or without geneticin (G418, 100 μg·ml⁻¹; Biochrom), in 5% CO₂ at 37°C. Cells were passaged 1:3–1:20 every second to third day from P8 to P28 depending on confluence; 24 hr after seeding, confluent HEK WT cells (70–90%) were incubated with 1 μg per 200‐μl transfection mix of each plasmid containing the different cDNAs using X‐treme GENE HP DNA (Roche, Mannheim, Germany) reagent following the supplier's recommendations. For stable transfection, pcDNA™3.1 carrying the cDNA of MOR‐H297^6.52A was linearized with restriction enzyme BglII (NEB, Frankfurt am Main, Germany). After 48 hr, the medium containing the transfection reagent was removed and geneticin (G418; 100 μg·ml⁻¹) was added with standard culture medium for selection of successfully transfected cells. Membrane expression of the transfected receptors was qualitatively verified via immunocytochemistry (data not shown). Stably transfected, polyclonal cell lines were cultured for a maximum of 23 passages.

Yeast genomic DNA was isolated from 1 x 10⁹ cells using Genomic-tip 20/G (QIAGEN) as described in [54 (link)]. After digestion with the BglII restriction enzyme (New England Biolabs) half of the purified DNA was subjected to Neutral-Neutral two-dimensional gel analysis as described in [55 (link)]. Southern blotting was carried out with the probes shown in Fig 2C, which were generated by PCR using genomic DNA as template. Probe 1 was generated as described above. For probe 2, recognizing the BglIIA fragment 5’-GTTTCTTTTCCTCCGCTT-3’ and 5’-ATCTCTTGGTTCTCGCAT-3’ were used as forward and reverse primers, respectively. For the probe near TER102 on chr. I 5’-GAAGGTTCAACATCAATTGATTGATTCTGCCGCCATGATC-3’ and 5’- GCTTCCCTAGAACCTTCTTATGTTTTACATGCGCTGGGTA-3’ were used as forward and reverse primers, respectively.
For Neutral-Alkaline two-dimensional gel electrophoresis BglII digested DNA was run on a Neutral gel in the first dimension. In the second dimension the excised DNA was run on a 1.5% agarose gel in 50 mM NaOH plus 1 mM EDTA at 4°C [7 (link)]. Probe 3 (Fig 2C) used for southern blotting was made by PCR with 5’-CAGCCATAAGACCCCATC-3’ and 5’-GCAGTTGGACGTGGGTTA-3’ as forward and reverse primers, respectively, and genomic DNA as template.