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Figure 4


Fig. 4. EMSA and ChIP of Pou4f2-binding to site A within the Eomes retinal enhancer. (A) Pou4f2 binds to site A in vitro. A probe containing Pou4f2-binding site A forms a Pou4f2-DNA complex. The left-most lane shows the free probe (F.P.). The second lane shows the Pou4f2-DNA complex formed in the presence of in vitro-synthesized Pou4f2. Free probe and complex are indicated by arrows. The remaining lanes show the extent of complex formation in the presence of a 500-fold molar excess of wild-type (WT) and mutated (MT) site A oligonucleotides, site B oligonucleotide, and anti-Pou4f2 antibody. (B) ChIP analysis using retinas from E15.5 embryos shows occupancy of Pou4f2 at site A. Sequences around Eomes site A or Titin were amplified after immunoprecipitation with anti-Pou4f2/Brn3b, anti-acetylated-histone H3 or anti-histone H3 antibodies. The left lanes show the expected size of the amplified products generated using input chromatin extracts. The right lanes show the amplified products resulting from the immunoprecipitation of chromatin extracts. The middle lanes show the amplified products generated using mock immunoprecipitation of chromatin extracts without the primary antibody.





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