Skip to main content
. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Nat Cell Biol. 2011 Aug 14;13(10):1244–1251. doi: 10.1038/ncb2313

Figure 2.

Figure 2

Notch1 Negatively Regulates Active β-Catenin in ESCs and Physically Interacts with β-Catenin. a, Relative expression of β-Catenin and Cyclin D1 mRNA in ESCs transfected with control or Notch1 siRNA (100 nM), determined by qPCR. b, Western analysis of ESCs transfected with control or Notch1 intracellular domain (N1ICD) (100 or 300 ng) and cultured with BIO. GAPDH antibody was used as a loading control. c, Relative β-Catenin/TCF luciferase activity of BIO-treated ESCs transfected with control or N1ICD +/− MAML or RBP-J siRNA. d, Relative β-Catenin/TCF luciferase activity of ESCs transfected with control or Notch1 siRNA and cultured with or without BIO e, f, Transverse sections of control, IslCre;β-catenin(ex3)loxP (Act-β-Cat) or IslCre;Gt(ROSA)26Sortm1(Notch1)Dam/J (Act-β-Cat; N1ICD overexpression) embryos at embryonic day 9.5, stained with H&E (e) or β-Catenin antibody (red, f). Asterisks indicate precardiac mesoderm containing cardiac progenitor cells (e). Scale bars, 100 μm (e) or 25 μm (f). DAPI (blue) was used to counterstain the nuclei (f). nt, neural tube; ot, outflow tract; pe, pharyngeal endoderm; ec pharyngeal ectoderm; pm, precardiac mesoderm. g, h, ESCs treated with or without BIO (g) or SW480 (human colon cancer) cells (g, h) were transfected with expression constructs for Myc (−) or N1ICD-Myc (+), immunoprecipitated (IP) with anti-Myc antibody and immunoblotted (IB) with β-Catenin antibody recognizing its C-terminus (g), dephosphorylated (active) form, or the phosphorylated N-terminus (h). Notch expression was detected with anti-Myc antibody (g). i, Schematic representation of Notch1 deletion constructs and their interactions with β-Catenin. j, Co-IP of BIO-treated ESCs with Notch1 deletion constructs shown in (i) using antibodies indicated. Arrowheads indicate Notch1 expression. k, Relative β-Catenin/TCF activity of BIO-treated ESCs transfected with Notch constructs shown in (i). TM (transmembrane domain), R (RAM domain), ANK (Ankyrin repeats), TA (transactivation domain), P (PEST domain). BIO was used at 2 μM. All qPCR or luciferase values were normalized to Gapdh or Renilla activity, respectively. (mean± s. d.; n = 4; *P < 0.01). P values were determined using two-tailed Student’s t-test, type II (see Methods). Numbers on Western blots correspond to relative quantification. Con, control; N1KD, Notch1 siRNA.