Figure_6.tif (1.14 MB)

The DS1C/A site has a 3.5-fold increased affinity for LIP compared to the DS1 wild-type C/EBP site.

Download (0 kB)
figure
posted on 27.08.2012 by Shruthi Ravimohan, Lucio Gama, Elizabeth L. Engle, M. Christine Zink, Janice E. Clements

(A) Competition EMSA using nuclear extract obtained from HEK-293T cells transfected with the pCMV-Flag-LIP construct and incubated with the labeled DS1 WT oligonucleotide alone (lane 1); 10 to 1000-fold molar excess (X) unlabeled DS1C/A oligonucleotide (lanes 2–11); or labeled DS1 WT oligonucleotide and anti-FLAG antibody (lane 12). (B) Competition EMSA using nuclear extract obtained from HEK-293T cells transfected with the pCMV-Flag-LIP construct and incubated with labeled DS1C/A oligonucleotide alone (lane 1); 10 to 1000-fold molar excess (X) unlabeled DS1 WT oligonucleotide (lanes 2–11); or labeled DS1C/A oligonucleotide and anti-FLAG antibody (lane 12). (C) Concentration of unlabeled oligonucleotide (-log[nM]) versus the percentage (%) of Flag-LIP bound to the labeled oligonucleotide, as determined by densitometric analysis of band intensities from the competitive EMSAs (A and B), was plotted. A non-linear regression curve was then used to determine KD of Flag-LIP for the DS1C/A and DS1 WT C/EBP sites from the logEC50 (half maximal protein binding) values. Shown are representative EMSAs (n = 2).

History

Licence

Exports