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. 2011 Mar 18;6(3):e17957. doi: 10.1371/journal.pone.0017957

Figure 5. The Notch-2 promoter is transcriptionally activated via Lef-1/Tcf-4/ß-catenin mediated signaling.

Figure 5

(A) HCT116 cells were co-transfected with pGL3-TATA carrying N2PR −2327/−99 (or empty pGL3-TATA) and HA-S33Y-β-catenin in pCGN, hTcf-4 or mLef-1 in pcDNA6 as well as pSV-β-galactosidase control vector (n = 6). (B) HCT116 cells were co-transfected with pGL3-TATA carrying N2PR −110 (white columns) or mutated N2PR −110 SpeI (black columns) (or empty pGL3-TATA) and HA-S33Y-β-catenin in pCGN, hTcf-4 or mLef-1 in pcDNA6 as well as pSV-β-galactosidase control vector. Luciferase activity was normalized against pGL3-TATA background activity by dividing N2PR −2327/−99-pGL3-TATA relative expression with the relative expression from empty luciferase vector (n = 9). Error bars describe SEM. (C) ChIP in HCT116 cells co-transfected with His-tagged Lef-1 or Tcf-4, N2PR −2327/−99 and N2PR −110; immunoprecipitation with anti-His (6× His tag® antibody) or IgG control, PCR with primers encompassing LEF-1/TCF-site −110 in the Notch-2 promoter. A 172-bp segment of NCAPD2 promoter was used as a negative control. (D) Western blots with anti-His (6× His tag® antibody) verifying the transfection of His-tagged Lef-1 (∼65 kDa) and Tcf-4 (∼45 kDa) in HCT116. Adjustments in whole image contrast levels were performed in Adobe Photoshop CS4.