Figure 4

The effect of BEX2 expression on the phosphorylation of p65, IκBα, c-Jun, and JNK Kinase activity. (A) Phospho-p65 level using ELISA. The levels of phospho-p65 (Ser468) and total-p65 were measured using ELISA after transfections with either control-siRNA or BEX2-siRNA in MCF-7 cells. Relative ratio for phospho-p65/total-p65 is shown for each group. *, is for BEX2-KD vs control experiments. Error Bars: ± 2SEM. (B) Phospho-IкBα level by western blot analysis. The levels of phospho-IкBα and total-IкBα were measured by western blot analysis after transfections with either control-siRNA (CT) or BEX2-siRNA (KD) in MCF-7 cells. IκB-α rabbit polyclonal and phospho-IκB-α (Ser32/36) mouse monoclonal antibodies were used at 1:1000 dilutions. Rabbit polyclonal α-tubulin was used as the loading control. Fold changes (RR) in band densities following BEX2-KD were measured relative to the control group (CT-siRNA). (C) Measurement of p65 DNA binding in MCF-7 cells using ELISA. The measurements were carried out in the following groups; 1) control: control-siRNA, 2) C2: control-siRNA + ceramide treatment at 10 μM ON, 3) C2/BEX-KD: BEX2-siRNA + ceramide, 4) BAY11: control-vector + BAY11 at 5 μM ON, and 5) BAY11/BEX2+: BEX2-vector + BAY11. *, is for ceramide or BAY11 group vs control; **, is for ceramide group vs ceramide + BEX2-KD. Error Bars: ± 2SEM. (D) Phospho-c-Jun level by western blot analysis. The levels of phospho-c-Jun (Ser63) and total-c-Jun were measured by western blot analysis after transfections with either control-siRNA (CT) or BEX2-siRNA (KD) in MCF-7 and MDA-MB-231 cell lines. Total-c-Jun rabbit monoclonal and phospho-c-Jun (Ser63) rabbit monoclonal antibodies were used at 1:1000 dilutions. Fold changes (RR) in band densities following BEX2-KD were measured relative to the control group (CT-siRNA). (E) JNK Kinase activity. JNK kinase assay was carried out using a selective immunoprecipitation of JNK followed by JNK kinase assay and western blot for phospho-c-Jun (Ser63). JNK kinase activities were measured after transfections with either control-siRNA (CT) or BEX2-siRNA (KD) in MCF-7 cells. Ceramide (C2) treatment at 10 μM overnight was used as a positive control for JNK induction. Fold changes (RR) in band densities following ceramide treatment and BEX2-KD were measured relative to the control group (CT-siRNA).