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. 2012 Oct 31;7(10):e47803. doi: 10.1371/journal.pone.0047803

Figure 1. Regulation of Bcl-2 and Bcl-XL expression by RanBPM.

Figure 1

(A) Down-regulation of RanBPM leads to enhanced Bcl-XL 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-XL, β-actin and RanBPM. (B) RanBPM shRNA cells exhibit enhanced Bcl-2 and Bcl-XL 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-XL, specific primers. Relative quantification of Bcl-2 and Bcl-XL gene expression was determined using the ΔΔC(t) method with Bcl-2 and Bcl-XL 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). *, P<0.05. Inset, 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. (C) 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. (D) 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.