Skip to main content
. Author manuscript; available in PMC: 2011 Sep 29.
Published in final edited form as: J Invest Dermatol. 2011 May 19;131(9):1877–1885. doi: 10.1038/jid.2011.142

Figure 2.

Figure 2

Differential binding of Slug, Snail and Twist to E-boxes of the ZEB1 promoter. Four Cy3- labeled 20nt wild type (wt) fragments of the ZEB1 promoter (wt-Cy3), each containing one out of four potential Slug binding sites (E-boxes) found in the ZEB1 promoter in a region from −3000 to +200 relative to the transcription start were incubated with 10μg of nuclear extracts of WM164 (a) or WM9 (b) in the absence or presence of a 20-fold molar excess cold probe (20 x wt unlabeled) for 30min at 37°C in binding buffer. DNA-protein complexes were separated by non denaturating PAGE (6%) for 45min, 120V and detected by Bio-Rad Molecular Imager FX (EMSA-Gel). After detection, complexes where transferred to PVDF membrane and subjected to immunoblotting with antibodies against Slug, Snail and Twist. To prove the specificity of the binding of EMT regulators to the contributing E-boxes, wild type sequences CASSTG were mutated to ATSSTA (mut-Cy3).