For protein extraction cells were homogenized in lysis buffer containing 50 mM Tris-Cl pH7.6, 150 mM NaCl, 1% (v/v) Triton 6100, 5 mM EDTA pH 8.0, 1 mM PMSF, 1:25 (v/v), Protease Inhibitor (Roche Applied Science, 11836153001), 2 mM Na 3 VO 4 , 5 mM NaF, 2 mM Sodium Pyrophosphate, 13.3 mM b-Glycerophosphate disodium salt. Homogenized cells were centrifuged at 13.000 rpm for 15 min at 4uC and protein samples were stored at 220uC until use. For detection of pSmad1/5/8 separated proteins were transferred to Immobilon-P membrane (Millipore, IPVH00010) and analyzed using rabbit anti-pSmad1/ 5/8 (Chemicon, ab3848, 1:1000) coupled with the anti-rabbit HRP (Santa Cruz SC2301, 1:5.000) and anti-b actin (Sigma-Aldrich, A5316, 1:5.000) combined with the anti-mouse HRP (Sigma-Aldrich, A4416, 1:40.000). Densitometry was done using the ImageJ 1.41 software.