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. 2013 Jun 20;8(6):e66892. doi: 10.1371/journal.pone.0066892

Figure 3. AP-1 binds directly to the PAK3 promoter at (+52/+60) both in vivo and in vitro.

Figure 3

A: Electrophoretic Mobility Shift Assay (EMSA) showing protein binding to the (+52/+60) PAK3 promoter region. Lane 1 shows the presence of a DNA/protein complex when the (+52/+60) PAK3 promoter radioactively-labelled oligomer was incubated with protein cell lysate of Rat1a-J4 cells treated with doxycycline. This DNA/protein complex could be competed for with unlabelled (+52/+60) wild-type (WT) oligomers (lane 2), but not with a mutated (+52/+60) sequence oligomer (lane 3). The second panel shows supershifted complexes using anti-cJun (lane 4), -JunB (lane 5) and –JunD (lane 6) antibodies. B: Chromatin Immunoprecipitation Assay (ChIP) showing cJun binding to the (+52/+60) PAK3 promoter region. Rat1a-J4 cells were treated with doxycycline for 48 hrs, after which DNA and protein complexes were cross-linked and pulled down with an anti-cJun antibody. RT-PCR amplification off the pulled-down chromatin showed cJun binding to the (+52/+60) PAK3 promoter region in the presence of doxycycline.