Mouse EOUs were cultured for 2 days with or without NOG inhibitor. Mouse TOUs were cultured for 10 days with or without NOG. (A) Protein was collected at the end of culture and Western blots were run for pSMADs 1/5/9 and pSMADs 2/3. (B) Quantification of Western blot intensity using Image J. (C) To confirm that the activity of NOG inhibitor was indeed specific to NOG, we added NOG and NOG inhibitor together to the culture to examine whether the effects of NOG inhibitor on phosphorylated SMAD 1/5/9 were blocked. Experiments were done with mEOUs (6 different litters). Phosphorylated SMAD protein was normalized to total SMAD protein and VDAC was used as loading controls. (D) mEOUs were cultured for 10 days and IHC was used to test for total or phosphorylated SMAD 1/5/9 expression. All images were obtained at ×20. Scale bar: 100 μm. Fluorescence intensity of total SMAD1/5/9 and pSMAD1/5/9 was evaluated using Image J (n = 3–6), *P < 0.05, **P < 0.005, ***P < 0.001.