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

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posted on 27.08.2012, 01:48 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).

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