Figure 5

The effect of BEX2 expression in c-Jun stable lines. (A) The levels of c-Jun and cyclin D1 in c-Jun stable clones. Stable c-Jun(+) clones (clones 1 and 2) were generated following the transfection of MCF-7 with c-Jun/pcDNA3.1 vector followed by selection in the presence of Geneticin. Transfection with an empty pcDNA vector was used as a control (vector). Western blot analysis for c-Jun and cyclin D1 were carried out using c-Jun and cyclin D1 rabbit monoclonal antibodies at 1:1000 dilutions. Rabbit polyclonal α-tubulin was used as the loading control. Fold changes (RR) in band densities for c-Jun(+) clones 1 and 2 were measured relative to the vector control. (B) The levels of cyclin D1 following BEX2 knock-down. The levels of cyclin D1 was measured by western blot analysis after transfections with either control-siRNA (CT) or BEX2-siRNA (KD) in c-Jun(+) stable clones. Fold changes (RR) in band densities following BEX2-KD were measured relative to the control group. (C) The effect of BEX2 knock-down (KD) on cell proliferation using MTT assay. BEX2-KD was carried out in stable c-Jun(+) clones and vector control (CTL). Absorbance measurements at 570 nM are demonstrated for BEX2-KD and control-siRNA experiments in the stable lines. *, is for clone 1 or 2 vs control vector and **, is for BEX2-KD vs control-siRNA. Error Bars: ± 2SEM. (D) BEX2 regulation of PP2A activity. PP2A immunoprecipitation phosphatase assay in c-Jun(+) clones 1 and 2 after transfections with either control-siRNA (CT) or BEX2-siRNA (KD). Pmoles of phosphate are demonstrated for each group. *, is for BEX2-KD vs control-siRNA. Error Bars: ± 2SEM.