Figure 3

Analyses of an in vivo binding of c-Jun (or phosphorylated c-Jun) to the miR-21 upstream promoter/enhancer region in MDA-MB-468 cells. In order to analyze the interaction between c-Jun/phospho-c-Jun and the upstream promoter/enhancer region of miR-21 promoter in MDA-MB-468 cells, ChIP assay was performed in MDA-MB-468 cells following protocols described in Materials and methods using the AP1 binding site containing miR-21 promoter (upstream promoter/enhancer region)-specific primers by PCR. Identical volumes from the final precipitated materials were used for the PCR reactions [untreated cells (A: lane 1); cells treated with HA for 1 h (A: lane 2); cells pretreated with anti-CD44 antibody plus 1 h HA addition (A: lane 3); cells treated with non-immune rat IgG (without HA addition) (A: lane 4) or cells treated with non-immune rat IgG plus 1 h HA addition (A: lane 5); or cells treated with vehicle control plus 1h HA addition (B: lane 1) or treated with JNK inhibitor plus 1h HA addition (B: lane 2); or cells pretreated with scrambled siRNA plus 1h HA addition (C: lane 1); or cells pretreated with c-Jun siRNA plus 1h HA addition (C: lane 2)]. (a: anti-c-Jun-mediated immunoprecipitated material; b: anti-phospho-c-Jun[pS63]-mediated immunoprecipitated material; c: IgG isotype control-mediated precipitated material; d: total input materials). [The ratio of c-Jun IP materials (a) or phosphorylated c-Jun IP materials (b) or IgG IP materials (c) and total Input materials (the loading control) (d) was determined by densitometry, and the levels were normalized to untreated (without HA treatment) value or non-immune IgG (without HA treatment) (designated as 1.00); the values expressed represent an average of triplicate determination of 4 experiments with an SD of less than 5%]. ND represents Not Detectable.