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. 2004 Feb 19;23(5):1155–1165. doi: 10.1038/sj.emboj.7600069

Figure 3.

Figure 3

Activation of the Notch receptor is not involved in TGF-β induction of HEY1. (A) Histograms show the relative abundance of HEY1 mRNA expression as determined by quantitative RT-PCR in keratinocytes treated with TGF-β in the absence or presence of γ-secretase inhibitor GSI. (B) Immunoblot showing NICD in nuclear and cytoplasmic protein fractions of keratinocytes treated with TGF-β. PARP (poly-α-ADP-ribosyltransferase) and TUBB (tubulin β) demonstrate purity of cytoplasmic and nuclear protein lysate fractions, respectively. (C) EMSA demonstrates binding of CBF-1 protein complex with oligonucleotide probe containing the distal CBE of the HEY1 promoter (lanes 1–5). Lanes 6 and 7: EMSA using mutated CBE oligonucleotide probe (CBEmut) with single bp exchange (cgtggGaaa → cgtggCaaa). Lanes 8 and 9: EMSA using probe containing the distal (−3005 bp) HEY1 SCR sequence (HEY1 distal SCR) demonstrates TGF-β-inducible Smad protein complexes.