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

Figure 4. RanBPM inhibits ERK1/2 activation through regulation of c-Raf.

Figure 4

(A) RanBPM regulates ERK1/2 signaling downstream of Ras. RanBPM shRNA Hela cells were left untransfected, or were transfected with either constitutively active RasV12 and RanBPM si-mt or RasV12 and empty pCMV vector. 24 h post-transfection, whole cell extracts were prepared and analyzed by western blotting. MEK1/2 and ERK1/2 activation was assessed by hybridization with phospho-MEK1/2 and phospho-ERK1/2 antibodies respectively and total MEK1/2 and total ERK1/2 levels were assessed using MEK1/2 and ERK1/2 antibodies. Expression of RasV12 and RanBPM was determined with an HA antibody. (B) RanBPM expression down-regulates c-Raf protein levels. RanBPM shRNA Hela cells were left untransfected, or were transfected with either constitutively active c-Raf (pEBG-GST-ΔN-c-Raf) and empty pCMV vector, or GST-ΔN-c-Raf and RanBPM si-mt. 48 h post-transfection, whole cell extracts were prepared and analyzed by western blotting. c-Raf expression was determined using a GST antibody, and ERK1/2 activation was assessed using a phospho-ERK1/2 antibody. RanBPM expression was verified using an HA antibody, and γ-tubulin was used as a loading control. (C) RanBPM shRNA Hela cells were either left untransfected or were transfected with constitutively active c-Raf (pCMV-Flag-Y/Y-c-Raf) and 48 h post-transfection, whole cell extracts were prepared as in B. Expression of c-Raf was assessed using a Flag antibody, and RanBPM expression was determined using an HA antibody. GAPDH was used as a loading control. (D) Whole cell extracts were prepared from Hela and HEK293 control shRNA and RanBPM shRNA cells and endogenous protein levels were analyzed by western blotting with c-Raf and RanBPM antibodies, with β-actin used as a loading control. (E) Control and RanBPM shRNA Hela cells were either left untransfected, or were transfected with empty vector or RanBPM si-mt. 48 h post-transfection, whole cell extracts were prepared and analyzed as in D. (F) RanBPM down-regulation does not affect c-Raf mRNA levels. cDNA from Hela control and RanBPM shRNA cells was analyzed by qRT-PCR using specific primers for GAPDH and c-Raf. Gene expression was quantified using the ΔΔC(t), with c-Raf expression normalized to GAPDH. Expression in RanBPM shRNA cells was calculated relative to that of control shRNA cells (set to an arbitrary value of 1). Data represents the mean of nine independent experiments, with error bars indicating standard error (SE).