TGF-Beta Induced Erk Phosphorylation of Smad Linker Region Regulates Smad Signaling
Figure 2
Erk is activated in fibroblasts via the PI3K/c-Raf/MEK pathway.
(A) AKR-2B fibroblasts were treated with the PI3K inhibitor LY294002 or MEK inhibitor U0126 (10 µM) 30 minutes prior to addition of TGF-β (2 ng/ml) for 2 h. Cell lysates were probed with an antibody specific to phospho-Erk, blots were then stripped and reprobed for total Erk as a loading control. (B) AKR-2B fibroblasts were treated with TGF-β (2 ng/ml) for the indicated times. Cells were also treated with LY294002 (10 µM) 30 minutes before TGF-β was added for 2 h. Blots were probed using an antibody specific to phospho-c-Raf (Ser338), and total Erk as a loading control. The loading control blot was obtained using the lower molecular mass portion of the same blot. (C) AKR-2B fibroblasts were treated with LY294002, U0126 or Ras inhibitor FPTII (10 µM or 20 µM, respecitively) 30 minutes prior to addition of TGF-β (2 ng/ml) for 2 h. Cell lysates were probed with antibodies specific to phospho-Erk or phospho-Akt (S473) with the blots stripped and reprobed for the corresponding total protein as a loading control. Comparisons of the relative intensity of bands of Phopho-Erk relative to total Erk loading control was expressed as fold increase relative to untreated control (set at 1). Statistical analysis was used to determine if treatments were not significant (NS), or significant at P<0.05 (*) or P<0.01 (**). Analysis was performed on four independent blots and the mean values (±SEM) shown.