Fig. 3. Evaluation of Smad and JNK signaling in MTEC exposed to TGF-β1. (A) Assessment of Smad2 phosphorylation and Smad4 nuclear localization in MTEC in response to TGF-β1. Phospho-Smad2 and Smad4 were analyzed by western blot of nuclear extracts, after 120 or 240 minutes of exposure to 5 ng/ml TGF-β1. Note that samples were run on the same gels, but irrelevant lanes were deleted for consistency of presentation. (B) Electrophoretic mobility shift assay evaluating binding of nuclear proteins to the consensus SBE in response to TGF-β1 (10 ng/ml) for the indicated times. (C) Assessment of phosphorylation of JNK1 and JNK2 following stimulation of MTEC with TGF-β1 (5 ng/ml) for the indicated times. The upper band indicates predominantly JNK2, whereas the lower band mainly reflects JNK1. Fold change denotes differences in signal compared to sham controls based upon densitometric evaluation. The lower panel represents total JNK1 and 2 western blot as loading control. Duplicate lanes in panels A, B and C represent results from independent samples.