Kaminska Bozena
Laboratory of Transcription Regulation, Nencki Institute, Warsaw, Poland.
Methods Mol Biol. 2009;512:249-64. doi: 10.1007/978-1-60327-530-9_13.
Tumor cells recruit inflammatory cells to the tumor site and transform them into tumor-supportive cells which in turn release numerous cytokines, including Transforming Growth Factor-beta that enhances tumor proliferation, invasion, angiogenesis and induces immune paralysis. Activation of JNK/c-Jun signaling pathway by various stimuli often leads to a formation of the AP-1 transcriptional complex, which is a critical regulator of a complex program of gene expression that defines the invasive phenotype. Recent studies on JNK/c-Jun phosphorylation have been carried out using phospho-specific antibodies, which have greatly facilitated analysis of signal transduction. The electrophoretic mobility shift assay (EMSA, gel shift) helps in determining the transcription factor activation and is based on the observation that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments or double-stranded oligonucleotides. The specificity of the DNA-binding protein is established by competition experiments and the protein composition of DNA binding activity can be analyzed with specific antibodies in a supershift assay. EMSA provides a sensitive and quantitative measure of a particular DNA binding activity under various experimental conditions.
