Luciferase assays were performed in 12 well format by transfecting 0.6 µg pGL3ti-(SBE)4 or pAR3-Luc, 0.012 µg pCH110 with or without 0.6 µg TβRI or 0.6 µg Smad 2-HA and 0.006 µg Smad4-HA, 40.0 pmoles siRNA duplex per well using Lipofectamine 2000 (Invitrogen). In experiments using TGF-β1 (R&D Systems) or Activin-A (R&D Systems) cells were allowed to grow for 48 hours in 10% FBS, DMEM, 2 mM glutamine prior to adding TGF-β1 at 5 ng/ml or Activin-A at 30 ng/ml. Cells were induced for 24 hours then lysed with 250 µl siGlow Lysis Buffer (Promega) according to manufacturer's procedures. Transfections utilizing the constitutively active receptors were incubated for 48 hours prior to lysis as above. 50 µl or 100 µl of lysate was assayed with 100 µl Bright-Glow Luciferase Assay Reagent (Promega). Transfection efficiency was normalized to β-galactosidase levels by assaying 10 µl lysate to 100 µl Beta-Glo Assay Reagent (Promega). Relative Luciferase units were obtained by dividing the luciferase activity levels by the β-galactosidase levels. Experiments were repeated at least twice and yielded essentially the same results.
