Journal: PLoS ONE
Article Title: p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts
doi: 10.1371/journal.pone.0066892
Figure Lengend Snippet: 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.
Article Snippet: Lysates was then diluted with dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-Cl, pH 8.1, 1 X Complete Protease Inhibitor) and chromatin was either immunoprecipitated with or without anti-cJun antibody (sc-44 (Santa Cruz)) and treated with pre-blocked protein-A agarose beads (Merck).
Techniques: Electrophoretic Mobility Shift Assay, Protein Binding, Incubation, Sequencing, Chromatin Immunoprecipitation, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Amplification