Geneticine

The mammalian expression vector, pcDNA3, CHO-K1 suspension (CHO-S) cells,
FreeStyle MAX reagent, FreeStyle CHO expression medium, pCMV-ARMS1-PK2
expression vector, antibiotics, and AssayComplete medium were purchased from
Invitrogen Corporation (San Diego, CA, USA). The following reagents and
materials were also used: restriction enzymes and a DNA ligation kit (Takara,
Tokyo, Japan); CHO cells (Japanese Cancer Research Resources Bank, Tokyo,
Japan); and Ham's F-12 medium, Opti-MEM I, serum-free CHO-S-SFM II,
Geneticine, and Lipofectamine 2000 (Gibco BRL, Grand Island, NY, USA). Fetal
bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT, USA).
Pro-Prep^TM protein-extraction solution was obtained from Intron
Biotechnology (Seoul, Korea). Lumi-Light western blot kit was purchased from
Roche (Basel, Switzerland). PathHunter CHO-K1 β-arrestin Parental cell
line was obtained from DiscoveRx (San Diego, CA, USA). Disposable spinner flasks
and grass flasks were obtained from Corning Incorporated (NY, USA). The cAMP
Dynamic 2 immunoassay kit was purchased from Cisbio Bioassay (Codolet, France).
All other reagents used were obtained from Sigma-Aldrich Corp (St. Louis, MO,
USA). The oligonucleotides used in this study were synthesized by Genotech
(Daejon, Korea).

B16.OVA, a mouse melanoma cell line expressing chicken ovalbumin (OVA) derived from C57BL/6J, was kindly provided by Prof. Vincenzo Cerullo. Cells were cultured according to ATCC recommendations in RPMI 1640 medium. Medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), penicillin (50 U/mL), streptomycin (500 μg/mL), and glutamine (4 mmol/L) in a humidified atmosphere with 5% CO₂ at 37°C. 1% of Geneticine (GIBCO) was added to the cells. At the endpoint of in vivo experiments, peripheral blood mononuclear cells (PBMCs) were isolated from the spleens of treated mice, to perform the IFN-γ ELISpot.