Fig. 2

Activin but not TGFβ utilizes PI3K/Akt to downregulate p21 in a SMAD4-independent manner. a Activin but not TGFβ leads to PI3K activation in a SMAD4-independent manner. ACVR2A/TGFBR2 wild type FET cells, FET with SMAD4 knockdown, and the SMAD4-null colon cancer cell line SW480 were stimulated with activin or TGFβ for 24 h following serum starvation. pAkt level was determined by Western Blot. Akt was used as a loading control. pAkt increased after cells were treated with activin but not TGFβ. b ACVR1B, ACVR2A’s primary binding partner, interacts with p85, the regulatory subunit of PI3K in an activin-dependent manner. Co-immunoprecipitation (Co-IP) with ACVR2A/TGFBR2 wild type FET cells were used to detect a protein-protein interaction between ACVR1B and p85. c p21 downregulation is dependent on Akt, a PI3K downstream target. ACVR2A/TGFBR2 wild type FET cells were transfected with siRNA to Akt1/2 (KD) and treated with activin or TGFβ for 24 h following serum starvation. Activin-induced downregulation of p21 was abrogated after Akt1/2 knockdown implicating Akt in activin-induced p21 regulation. d Knock down of downstream target in FET cell. ACVR2A/TGFBR2 wild type FET cells were transfected with siRNA Akt1/2 and siRNA SMAD4. Resulting loss of respective protein expression is shown using Western blotting. For siRNA SMAD4 we tested two different siRNA from Ambion (A: middle panel) and Santa Cruz (SC: right panel) and the latter was used in all our experiments. (C control; A Activin; T TGFβ; KD siRNA Akt1/2; IP immunoprecipitation)