P19 cells were obtained from the ATCC (ATCC Number CRL-1825). Cells were grown in 10% FBS, DMEM, 2 mM glutamine, penicillin and streptomycin. Cells were transfected using an Amaxa Nucleofector device using solution V and program C-20 according to manufacturer's procedures. 2.0×106 P19 cells were transfected with 400 nmoles siRNA duplex (Darmacon) or a GFP-Max nucleofection control. Post nucleofection 3.0×105 cells were plated to 12 well plates. Cells were allowed to grow for two days then were serum starved for 18 hours in 0.1% FBS, DMEM, 2 mM glutamine, penicillin and streptomycin. Cells were induced with 5 ng/ml TGF-β1 (R&D Systems) in low serum growth medium overnight. Cells were lysed using TriReagent reagent (Sigma) and RNA and protein were purified according to manufacturer's procedures.
