Fig. 5. EGF attenuates TGFβ1-mediated Smad2 C-terminal phosphorylation and Smad-driven promoter activity, but does not attenuate TGFβ1-mediated expression of TGIF, SnoN or cSki. (A,B) OKF6 cells were serum starved overnight and then pre-treated with EGF (20 ng/ml) for 30 minutes prior to the addition of TGFβ1 (10 ng/ml) for the indicated time points. The cell lysates were then probed for phosphorylated Smad2, and then stripped and re-probed for total Smad2. OKF6 cells were plated onto glass coverslips, serum starved overnight and then pre-treated with EGF (20 ng/ml) for 30 minutes, and then treated with TGFβ1 for 1-8 hours. The cells were then fixed in glutaraldehyde, permeabilized with Triton X-100, incubated with anti-Smad2 antibody followed by Alexa-Fluor-488-labeled secondary antibody. The immunofluorescence signal was detected using Zeiss microscope. (C) OKF6 cells were transfected with 1 µg of SBE-4 reporter luciferase construct, and then equal numbers of cells were plated overnight in 12-well tissue culture plates, serum starved for 8 hours and then pre-treated with EGF (20 ng/ml) for 30 minutes prior to treatment with TGFβ1 (10 ng/ml) for 24 hours. The cells were lysed, luciferase activity was quantified, normalized to untreated samples arbitrarily set at 1.0. ^**P<0.005, significantly different from EGF-,TGF-b1+ samples with. The results are representative of at least four independent experiments. (D) OKF6 cells were serum starved overnight and then treated with 20 ng/ml EGF 30 minutes prior to treatment with TGFβ1 (10 ng/ml) for 24 hours. The relative levels of TGIF, SnoN, cSki and GAPDH mRNA expression were determined by real time PCR, and normalized to untreated samples. The results are representative of at least two independent experiments.