Research Paper Volume 12, Issue 13 pp 13594—13617

Figure 7. Electrophoretic mobility shifting assays (EMSA) demonstrated that CREB directly binds to the promoter enhancer sequences of the αB-crystallin gene to control its expression. Bioinformatics analysis revealed that the mouse αB-crystallin gene contains many half CREB binding sites in the proximal promoter, and completely conserved CREB sites in the upstream enhancer or downstream enhancer regions (Supplementary Figure 3). EMSA revealed that CREB can directly bind to the conserved CREB binding sites in both upstream (M8) or downstream (M10). A-a, diagram of the two oligos containing a well-conserved CREB binding site (WT-M8, top) or mutant CREB binding site (MT-M8, bottom), which were used for gel mobility shifting assays described in A-b. A-b, gel mobility shifting assays. Nuclear extracts prepared from pCI-CREB-αTN4-1 cells were incubated with γ-32P-ATP-labeled oligo-nucleotide containing wild-type CREB binding site (A-a, top) under various conditions shown in the figure. B-a, diagram of the two oligos containing a less conserved CREB binding site (WT-M10, top) or mutant CREB binding site (MT-M10, bottom), which were used for gel mobility shifting assays described in B-b. B-b, gel mobility shifting assays. Nuclear extracts prepared from pCI-CREB-αTN4-1 cells were incubated with γ-³²P-ATP-labeled oligo-nucleotide containing wild-type M10 CREB binding site (B-a, top) under various conditions shown in the figure.