To simulate Smad signaling in a different way we co-transfected combinations of Smads 2,4 with constitutively active TβRI (ALK5), the type I receptor for the TGFβ signaling pathway (Figure S24C) [55]. Using this system we observed efficient reduction in the activation of the SBE regulated luciferase reporter gene with a siRNA to Bptf but not a mock siRNA control (Figure S24D). We repeated the experiments using multiple unique siRNAs and measured the transcription of endogenous TGFβ regulated genes. In these experiments we used three individual Bptf siRNAs which were effective in knocking down protein expression after 2 days of culture (Figure S24E). We then stimulated the P19 cells with TGF-β1. Like nodal and activin-A, TGF-β1 stimulates the phosphorylation of Smad 2/3 through the dimerization and activation of similar type I and II receptors. We similarly observed a significant reduction of Cer1 and T transcription in Bptf depleted cells upon Smad2/3 activation with TGF-β1 (Figure S24F). We also used the human breast cancer cell line MCF10CA1 in similar assays to determine if BPTF could play a role in Smad signaling in
