Figure 5

EpCAM expression is associated with phosphorylation of the AP-1 transcription factor protein subunit c-Jun. (a) Specific ablation of epithelial cell adhesion molecule (EpCAM) is associated with decreased c-Jun phosphorylation as measured by multiplex phosphoprotein assay. Stably transduced CA1a cells (scrambled control shRNA construct (SCR) and sh2) were induced with epidermal growth factor (EGF), and phosphorylation was measured using a multiplex phosphoprotein assay. Relative levels of phosphorylated (p)-AKT, p-c-Jun, and p-JNK were measured by flow cytometry and expressed as mean fluorescence intensity (MFI). Error bars represent standard error. (b) Specific ablation of EpCAM is associated with decreased c-Jun phosphorylation as measured by phosphoprotein immunoblot. Stably transduced CA1a and MCF-7 cells (SCR and sh2) were serum-starved for six hours. The cells were treated with 20 μM anisomycin for 15 minutes and 30 μg of the cell lysate was analyzed with antibodies recognizing EpCAM, superoxide dismutase (SOD, loading control), p-c-Jun-63, or total c-Jun. Relative band density was quantified using ImageJ software, with the results indicated below the relevant band. (c) Recombinant EpCAM is capable of rescuing c-Jun phosphorylation in CA1a(sh2) cells. CA1a(sh2) cells were serum-starved for six hours and then treated with rEpCAM at concentrations of 50, 100, and 200 ng/mL for two hours. The cells were then stimulated with anisomycin for 10 minutes. A 30 μg sample of each cell lysate was analyzed for p-c-Jun and total c-Jun. Relative band density was quantified using ImageJ software, with the results indicated. The results are representative of two independent experiments.