Detection of the Content and Activity of the Transcription Factor AP-1 in a Multistage Skin Carcinog

Investigation of transcription factor activity in animal tissue during the early stage of cancer development can be difficult because of a low number of affected cells in a background of a large number of normal cells. We have used a well-established multistage skin carcinogenesis model to study the effect of manganese superoxide dismutase on the activity of AP-1 during an early stage of mouse skin carcinogenesis. The DNA binding activity of AP-1 proteins to a concensus DNA regulatory binding element known as TRE (TPA Responsive Element) is used as an in vitro assay for AP-1 activity in the nucleus of skin cells. This activity is detected by electrophoretic mobility shift assay (EMSA) which is based on the ability of specific proteins to bind and retard the migration of radioactive labeled oligonucleotides on a native gel. The presence of a specific protein in the complex can be identified by adding to EMSA an antibody specific for that protein, which will result in supershift complexes on the gel. The presence of the proteins in question can be further verified by standard Western analysis of the nuclear proteins.