Cos 7

The African green monkey kidney-derived cell line, COS-7, was obtained from the American Type Culture Collection (CRL-1651) and cultured in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% fetal calf serum and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin) at 5% CO₂ and 95% air, maintained at 37 °C in a humidified incubator. Cells were transfected in 35-mm Petri dishes when the culture reached 50–60% confluence, with DNA (2 µg total DNA) complexed with jetPEI (Polyplus transfection) according to the standard protocol recommended by the manufacturer. COS-7 cells were co-transfected with 200 ng of pCI-SCN5A (NM_000335.4), 200 ng of pRC-SCN1B (NM_001037) (kind gifts of AL George, Northwestern University, Feinberg School of Medicine) and 1.6 µg pEGFP-N3 plasmid (Clontech). Cells were re-plated onto 35-mm Petri dishes the day after transfection for patch-clamp experiments. HEK293 cells stably expressing hNa_v1.5 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum, 1 mM pyruvic acid, 2 mM glutamine, 400 µg/ml of G418 (Sigma), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Grand Island, NY) at 5% CO₂ and 95% air, maintained at 37 °C in a humidified incubator.

COS-7 (African green monkey kidney) cells (Clontech, Mountain View, CA, USA) were cultured in 10 mL DMEM containing 10% (v/v) FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) on a 100-mm dish at 37 °C in a humidified 5% CO₂ and 95% air (v/v) atmosphere. Authentic OH-Cbl was added into the medium at various concentrations (0, 0.1, and 1.0 μmol/L) in the presence or absence of 1.0 μmol/L (Ade)OH-Cba. After reaching confluence, the cells were harvested and homogenized in 100 mmol/L potassium phosphate buffer (pH 7.0) containing leupeptin (1 mg/mL), aprotinin (10 mg/mL), and 0.1 mmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride, in a Teflon homogenizer at 4 °C. The homogenate was centrifuged at 10,000× g for 10 min at 4 °C, and the supernatant fraction was used as a crude enzyme solution for the enzyme assay.
HEK 293 (tet-on) (human embryonic kidney, tetracycline-induced expression system) cells were cultured in 10 mL DMEM containing FBS, penicillin, streptomycin, G418 (100 μg/mL), and NaHCO₃ (1 g/L) on a collagen-coated dish (100 mm) at 37 °C in a humidified 5% CO₂ and 95% air (v/v) atmosphere.