Figure 1.

Notch Negatively Regulates Active β-Catenin in Stem Cells Independently of RBP-J. a, Western analysis of ESCs transfected with control or Notch1 (N1) siRNA with active (Act), Phospho (Ser37), or total β-Catenin antibodies that detect N-terminal-dephosphorylated β-Catenin. b, c, Relative β-Catenin/TCF-directed luciferase activity in ESCs (b) or neural stem cells (NSCs) (c) transfected with control siRNA or siRNA against Notch1 or Notch1–4. β-Catenin/TCF activity was measured by co-transfecting cells with a luciferase reporter downstream of multiple TCF binding sites (Topflash). A mutant reporter (Fopflash) exhibited negligible activity in all luciferase assays done in this study. d, Relative RBP-J expression levels by qPCR in ESCs after transfection with control or RBP-J siRNA, determined by qPCR. e, Western analysis of ESCs transfected with control or RBP-J siRNA (50 or 100 nM) with Act β-Cat antibodies. f, Transverse sections of control, Notch1 knockout (KO) (Isl^Cre; Notch1^tm2Rko(ex3)^loxP) or RBP-J KO (Isl^Cr; RBP-J^flox/flox) embryos stained with H&E (top) or Isl1 antibody (green, bottom) at embryonic day 9.5, at level of outflow tract (ot). Asterisks indicate precardiac mesoderm containing cardiac progenitor cells. Dapi (blue) was used to counterstain the nuclei. The cutting plane is indicated by a dotted line (left). Scale bars, 100 μm. All luciferase values were normalized to Renilla activity (mean±s. d.; n = 4; *P < 0.01). P values were determined using two-tailed Student’s t-test, type II (see Methods). Gapdh antibody was used as a loading control. Numbers on Western blots correspond to relative quantification. h, head; ht, heart tube; Con, control; N1KD, Notch1 siRNA; N1–4KD, Notch 1–4 siRNA.