Fig. 4

Electrophoretic mobility shift assay (EMSA) using synthetic ESPL1 promoter-derived c-MYB binding site probes and Chromatin immunoprecipitation (ChIP). a Schematic drawing depicting the ESPL1 Separase promoter and location of predicted regulatory DNA motifs (drawing not to scale) (Pati 2008). The arrow shows the predicted transcription start site (TSS). Abbreviations: TATA, TATA box; PRE, progesterone responsive element; ERE, estrogen responsive element; p53, p53 binding element; c-MYB, predicted c-MYB binding element. Numbers denote upstream distances with respect to the TSS. b A FITC-labeled double stranded DNA oligonucleotide corresponding to the putative c-MYB binding site of the ESPL1 promoter was incubated with native BV-173 nuclear extract. DNA/protein complexes were resolved on a 0.5 % TBE 1.0 % native LE/GTG agarose gel. The left panel (tonal inversion) shows FITC-related fluorescence signaling of the gel before blotting, the right panel depicts the corresponding anti-c-MYB Western blot immunostaining. The lanes represent: lane 1, DNA target (FITC-labeled oligonucleotide) without nuclear extract; lane 2, DNA target with nuclear extract; lane 3, DNA target with nuclear extract and with 100fold molar excess of analogous unlabeled oligonucleotide as binding competitor. c ChIP analysis of BV-173 cells. DNA fragments immunoprecipitated by anti-c-MYB IgG and a non-binding control IgG were amplified by qRT-PCR. Results are expressed as percentage of input (average). ChIP results are derived from at least triplicate qRT-PCR measurements