TGF-Beta Induced Erk Phosphorylation of Smad Linker Region Regulates Smad Signaling
Figure 4
TGF-β directs Erk phosphorylation of Smad2 linker region.
(A) Western blots from AKR-2B fibroblast cell lysates treated with TGF-β (2 ng/ml) for the indicated time periods with or without U0126 (10 µM) for 120 min. Blots were probed for smad2 phosphorylated within the linker region at S245, 250 and 255, striped and reprobed for total smad2 to demonstrate each loading. (B) Receptor mediated phosphorylation of smad2 was also determined in the same samples using antibodies specific to C-terminal Ser 465/467. The blots were stripped and reprobed for total smad2 to demonstrate similar loading of all samples. Representative western-blots are shown with each time course performed in triplicate with consistent results. (C) Western blot and relative quantification of smad2 linker region phosphorylation in AKR-2B fibroblasts treated for 30 min. with or without TGF-β (2 ng/ml) or EGF (50 ng/ml). P-Erk blots are also shown to indicate Erk activation. Intensity of P-smad2 linker region band was determined and expressed graphically as fold increase (using total Erk band intensity as the loading control) relative to untreated control values for each experiment. The mean values of three independent experiments are shown (±SEM). Letters above each column indicate the different statistically significant (P>0.05) groupings. (D) Representative western blots showing phosphorylation of smad2 linker region of AKR-2B fibroblasts treated for 120 min. with or without TGF-β (2 ng/ml) and/or inhibitors U0126 or LY364947. Blots were stripped and reprobed for total smad 2 as a loading control.