For each of these genes, PCR signals for the expected fragment(s) containing the putative AP-1 binding site were obtained with chromatin precipitated with either an ATF3-specific or a JUN-specific antibody (Figure 6).
A mutated version (T789 m) of this construct carrying a 7-bp substitution in the miR-181 binding site was obtained through site-directed mutagenesis.
A deletion for the let-7e predicted binding site was obtained using the QuickChange II XL site-directed mutagenesis kit (Stratagene, Santa Clara, CA, USA).
Bioinformatics analysis found a potential binding site for KLF13 in porcine PPARγ promoter, while no binding site was obtained for KLF13 in mouse PPARγ2 promoter using same bioinformatics analysis method.
It is important to note that the structures of both human and bovine mincle CRDs in which the glutamic acid residue is displaced away from the primary binding site were obtained at low pH.
(1219) More-detailed information about structural requirements for binding and, indirectly, the geometry of the binding site was obtained by classification analysis, 1222) pharmacophore studies, pseudo-receptor modeling, 1225) GRIND descriptor studies, 1226) and 3D-QSAR analyses, which were combined with the homology models, based on the available bacterial ABC transporter structures.
Much more commonly a small collection of binding sites is obtained and from them a model for the TF's binding specificity is determined.
Regardless of how the collection of binding sites is obtained, and especially important for the use of motif discovery methods, a model for the TF's specificity must be used, and usually it is some form of PWM.
Collections of binding sites are obtained in simulation experiments where the true model for the transcription factor is known and various sampling procedures are employed.
