Regulation of Bcl-2 and Bcl-X_L expression by RanBPM.

<p>(<b>A</b>) Down-regulation of RanBPM leads to enhanced Bcl-X_L expression. Whole cell extracts were prepared from Hela and HCT116 control shRNA and RanBPM shRNA cells and were analyzed by western blotting. Blots were hybridized with antibodies against Bcl-X_L, β-actin and RanBPM. (<b>B</b>) RanBPM shRNA cells exhibit enhanced Bcl-2 and Bcl-X_L mRNA expression. cDNA from Hela control shRNA, RanBPM shRNA, and RanBPM shRNA cells re-expressing RanBPM via transient transfection of RanBPM si-mt construct was analyzed by qRT-PCR with RNA polymerase II (Pol II), Bcl-2, and Bcl-X_L, specific primers. Relative quantification of Bcl-2 and Bcl-X_L gene expression was determined using the ΔΔC(t) method with Bcl-2 and Bcl-X_L expression normalized to that of the controls. Bars represent values normalized to control shRNA cells. Data represents the mean of three independent experiments with error bars representing standard deviation (SD). *, <i>P</i><0.05. <i>Inset</i>, representative western blot analysis of whole cell extracts to control for the levels of RanBPM using a RanBPM antibody and β-actin as a loading control. (<b>C</b>) RanBPM expression down-regulates Bcl-2 protein levels. Hela RanBPM shRNA cells were transfected with pCMV-3xFlag-Bcl-2. 24 h post-transfection, cells were split and were either transfected with pCMV-HA-RanBPM si-mt or empty vector. Whole cell extracts were prepared 48 h later and analyzed by western blotting. Expression of ectopic Bcl-2 was determined by hybridization with anti-Flag antibody. RanBPM expression was assessed with a RanBPM antibody, and β-actin was used as a loading control. (<b>D</b>) Control experiment to confirm the specificity of RanBPM expression on Bcl-2 protein levels. This experiment was carried out the same as in C, except that RanBPM shRNA cells were transfected with pCGN-HA-ΔN-Oct-1 instead of Flag-Bcl-2. The truncated ΔN-Oct-1 migrates at 65 kDa as opposed to full-length Oct-1 (which migrates at 90 kDa), allowing for detection of Oct-1 and RanBPM expression in cells transfected with both constructs. Blots were hybridized with anti-HA antibody to verify Oct-1 expression.