Tcs401

Monoclonal rabbit-anti-human PTP1B was purchased from Abcam (Cambridge, UK). Monoclonal rabbit-anti-rat extracellular signal-regulated kinase (Erk)1/2, p-Erk1/2, protein kinase B (Akt; pan), and p-Akt were obtained from Cell Signaling Technology (Danvers, MA). Monoclonal mouse-anti-human α-smooth muscle actin (α-SMA), monoclonal rabbit-anti-human cyclin A and cyclin D1 antibodies, fluorescein isothiocyanate (FITC)-conjugated goat-ant-rabbit, and horseradish peroxidase-conjugated donkey-anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit-anti-human β-actin was obtained from Biomedical Technologies (Stoughton, MA). TCS-401 was obtained from Tocris Bioscience (Tocris, Bristol, UK). PD98059 (an inhibitor of mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/Erk)), LY294002 (an inhibitor of phosphatidylinositol 3-kinase (PI3K)), and propidium iodide (PI) were purchased from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Norcross, GA). BSA (BSA) was purchased from Fisher Scientific (Pittsburgh, PA).

All chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA), unless specified otherwise. CB₁R agonists, CB₁R antagonists, URB 597 and TCS 401 were purchased from Tocris (Bristol, UK). Recombinant leptin was purchased from Research and Diagnostic (R&D) Systems, dissolved in sterile 20 mM Tris-HCI (pH 8.0) and stored at -80°C in small aliquots. For intraperitoneal injection, leptin was diluted in isotonic sterile PBS or isotonic sterile NaCl to a concentration of 0.03 mg/mL. ACEA and URB 597 were diluted in a mixture of 90% sterile isotonic NaCl + 5% sterile DMSO + 5% Tween 80 to final concentrations of 0.2 mg/mL and 0.1 mg/mL, respectively. Injection volumes were limited to 10 μL/g of body weight.
If drugs were obtained in anhydrous ethanol, ethanol was first evaporated with N₂ and sterile dimethyl sulfoxide (DMSO) was then used for reconstitution. (DMSO was stored in small aliquots at -20°C to avoid water absorption into the solvent.) All stock solutions were split into small aliquots and a maximum of 2 freeze-thaw cycles were allowed. In cell culture experiments, final concentration of DMSO was limited to 0.15%.