In all cases, ��-actin was used as internal control to normalize Western blot data. Luciferase and ��-galactosidase assays. This procedure Intedanib was performed as described previously in detail (26). Briefly, 293T cells were grown in DMEM supplemented with 10% of fetal bovine serum, 100 U/ml penicillin, and 10 ��g/ml streptomycin. For the luciferase and ��-galactosidase assays, 293T cells were transfected with prepro-TRH-luciferase reporter, ��-galactosidase expression vector, and each plasmid (ObRb, STAT3, PKCR2-TR��2) or the empty expression vector. All transfections were performed with Mammalian Cell Transfection kit (Specialty Media, Phillipsburg, NJ), following the manufacturer’s instructions.

Twenty-eight hours after transfection, cells were incubated in serum-free Gibco OptiMEM (Invitrogen) and subsequently stimulated overnight with T3 (0, 10, and 100 nM) and/or further stimulation with leptin (0, 1, 10, and 100 nM) for 6 h. After stimulation, cells were lysed in lysis buffer A (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1% Triton X-100, and 2 mM dithiothreitol) and assayed for luciferase and ��-galactosidase activity. Enzymatic activities were measured using Luminometer (LB 9501; EG & G Berthold, Bad Wildbad, Germany). Luciferase activity was determined using Luciferin (Molecular Probes, Eugene, OR) and ��-galactosidase activity using Galacton (Tropix, Bedford, MA) as described by the manufacturers. Data show the relative luciferase activity, and it is the mean of at least three separate experiments performed in triplicate. Deiodinase activity assays.

Deiodinase activity assays were performed according to Richard et al. (48). Briefly, extracted BAT and liver tissue were homoginized in P100E2D1 and extracted, and ARC tissue was homoginized in P100E2D10. Total protein concentrations were determined using a Bradford assay. D2 activity in BAT and ARC samples was assayed using P100E2D25 and [3��5��-125I]T4 (3,000 counts/min) with 1 mM 6-propyl-2-thiouracil (PTU; Sigma p3755; to block D1 activity) and 1 ��m T3 (to block D3 activity) in a total reaction volume of 0.5 ml. D1 activity in liver samples was assayed using P100E2D10 and [3��5��-125I]T4 (3,000 counts/min) in a reaction volume of 0.5 ml. Liver samples were run with and without 1 mM PTU (a D1 inhibitor) to determine D1 activity only. D3 activity was assayed using P100E2D50 and [125I]T3 (3,000 counts/min) in a total reaction volume of 0.5 ml. Samples were incubated at 37��C for 30 min, and reactions were stopped with 0.2 ml ice-cold methanol. Samples were then centrifuged at 3,200 rpm for 30 min, and 0.5 ml of supernatant was Anacetrapib removed and counted for radioactivity. All samples were run in the same assay on the same day. Extracted ARC/ME tissue was homoginized in P100E2D10.