Tgf β1 antibody

Cell culture medium was purchased from Gibco BRL (BRL, Carlsbad, CA, USA). Culture dishes and plastic tubes were purchased from Corning (Corning, NY, USA). Cobalt chloride (CoCl₂) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Cyclohexylamine, genistein, monensin, TGF-β receptor I inhibitor (SB431542), and recombinant human TGF-β1 (rhTGF-β1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). P38 MAPK inhibitor (SB203580) was purchased from Calbiochem (Merck Chemicals, Gibbstown, NJ, USA). The TGF-β1 antibody was purchased from R&D Systems Inc. (Minneapolis, MN, USA).

Melanoma cell lines were obtained from ATCC and grown as monolayers in RPMI or DMEM media supplemented with 10% heat-inactivated fetal calf serum (FCS) in the presence of 5% CO₂ in a humidified atmosphere at 37°C. To guarantee cell line authenticity, B16F10 and COLO829 cell lines were used for a limited number of passages and routinely tested for the expression of melanocyte-lineage proteins such as MelanA/MART1. Human monocytic THP-1 cells obtained from Dr. A. Coste (University of Toulouse, France) were cultured in RPMI containing 10% FCS and 0.02 mM β-mercaptoethanol. THP-1 cells were differentiated into macrophages by stimulation with 20 ng/ml PMA (Sigma) for 24 hours; then cells were cultured for an additional 24 hours without PMA.
Conditioned media (CM) were produced by culturing melanoma cells in serum-free RPMI for 48 hours. In some experiments, melanoma cells were treated with 3 μM sphingosine kinase inhibitor SKI-I (5-(2-Naphthalenyl)-1H-pyrazole-3-carboxylic acid 2-[(2-hydroxy-1-naphthalenyl)methylene]hydrazide; Abcam). At the end of the culture period, the CM were collected, centrifuged for 5 min at 1500 rpm and filtered. Macrophages were then treated with the CM alone or in the presence of 5 μM S1P, 50 ng/ml recombinant TGF-β1 (Ebioscience SAS, Paris, France) or 1 μg/ml TGF-β1 antibody (Clone #1D11, R&D systems, Lille, France) for 48 hours.