Fig 6. DNA-Binding analysis of Ouib for the upstream element of spok.

(A) ABCD assay with nuclear extracts from S2 cells and avidin-biotin-conjugated double-stranded DNA probes. After pull-down, proteins were detected by western blotting using anti-FLAG antibody. (B) EMSA using recombinant proteins of GST alone or GST-fusion zinc finger domains of Ouib (amino acids 150–313) with ³²P-labeled double-stranded oligonucleotide probes containing the wild type 15 bp Ouib-response element (ORE). The x100 and x200 amounts of the non-labeled probes of the wild type ORE (ORE WT), the mutated ORE (ORE Mut) and the wild type M1BP-response element (MRE WT) were used as cold competitors. (C) Densitometric analysis of the EMSA band radioactive intensities in the same experimental condition as B with 3 independent replicates. Average radioactivity of the ORE/GST-Ouib-Zf complex is set as 100%. Note that the complexes with the ORE were outcompeted by the unlabeled ORE WT probe, but not by the unlabeled MRE WT probe. *P<0.05 and **P<0.01 by Tukey's multiple comparisons test. n.s., not significant. (D) EMSA using recombinant proteins of GST-fusion zinc finger domains of Ouib (amino acids 150–313) with ³²P-labeled double-stranded oligonucleotide probes of ORE WT, ORE Mut and MRE WT.