Figure 4. Smad1 is a competitor of the transcription factor TEAD in regulating mouse NSC proliferation. A, Mouse NSCs were transfected with 3xSD-luciferase reporter or BRE-luciferase plasmid and treated with 10 ng/ml BMP2 for different time periods. The luciferase reporter shows that BMP2 reduces YAP/TEAD-mediated transcriptional activity while enhancing Smad-mediated transactivation. B, Mouse NSCs were transfected with 3xSD-luciferase reporter and treated with BMP2. After 2 h, BMP2 was washed out and replaced with the normal medium. YAP/TEAD-mediated transactivity is gradually restored after withdrawal of BMP2 (t test; n = 3, p < 0.01). C, Flag-immunoprecipitates from NSCs transfected with Myc-YAP, WT, or Myc-YAP WW mutant plasmid together with Flag-Smad1 were immunoblotted with anti-Myc antibody and anti-Flag antibody. As a control, 2% of the input was blotted with anti-Flag or Myc antibody. D, Endogenous coimmunoprecipitation between YAP and Smad1/4 in murine embryonic NSCs shows that the YAP and Smad1/4 interaction is increased on BMP2 treatment. E, Myc-immunoprecipitates from 293T cells transfected Myc-YAP WT or Myc-YAP WW together with Flag-Smad1 or Flag-Smad1 Y227A were immunoblotted with anti-Flag antibody. Two percent input was blotted with anti-Flag, Myc, or GAPDH antibody. F, Murine NSCs were transfected with 3xSD-luciferase reporter and Myc-YAP, WT, or WW mutant plasmid. The luciferase assay shows that BMP2 inhibits WT but not WW mutant YAP-mediated transactivation (t test; n = 3, p < 0.01). BMP2 washout restores YAP-mediated transactivation. G, Representative images of neurospheres infected with retroviral YAP WT or YAP WW plasmids. Scale bar, 100 μm. H, I, Murine NSCs were infected with retroviral YAP WT or YAP WW expression vector. The number and size of the neurospheres formed after 1 week were counted and measured. YAP WW overexpression, not the YAP WT, could block the BMP2-induced proliferation inhibition (ANOVA; n ≥ 3, p < 0.01).