Fig. 3. TGF-ß downregulates INK4b expression in CEFs and chicken ovarian granulosa cells. (A) CEFs (35 PDs) were treated with 2 ng/ml of recombinant TGF-ß for the indicated times, and samples of total RNA were analysed for expression of INK4b. Equal loading was assessed by staining the ribosomal RNA bands with Methylene Blue. (B) Immunofluorescence detection of Smad2/3 in CEFs that were either untreated (upper panels) or treated with recombinant TGF-ß for 1 hour. (C) Cultured cGCs were transferred into medium containing 5% FCS but lacking additional growth factors for 16 hours. The cells were then treated with the indicated amounts of TGF-ß for 4 hours and samples of total RNA were analysed for expression of INK4b and ARF.