Fig. 5

Binding analysis of AP-1 recognition sequences from the human miR-21 promoter in cervical cancer cells. The AP1D, AP1M and AP1P probes were 5′-ends labeled with biotin and were incubated in absence (lanes 1–3) or in presence of nuclear extracts (NE; lanes 4–15), from SiHa (a), HeLa (b), C-33A (c) and HaCaT cells (d). The nuclear extracts were pre-incubated with 1000-fold molar excess of unlabeled AP1 probes as specific autologous competitor (AC; lanes 7–9) or with equimolar concentration of NF-kB probe as heterologous competitor (HC; lanes 10–12), before to add the labeled AP1 probes. In super-band shift assays, DNA–protein complexes were allowed to form prior to the addition of anti-c-Fos antibody (Anti-c-Fos; lanes 13–15). The arrows indicate the formation of specific retarded DNA–protein and super-band shift retarded complexes in each case, and free DNA is indicated. The data are representative of three independent experiments