Figure 5. RanBPM interacts with c-Raf and reduces c-Raf-Hsp90 association.

(A) Co-immunoprecipitation of RanBPM and c-Raf. RanBPM shRNA Hela cells were transfected with empty vector or RanBPM si-mt, and 48 post-transfection whole cell extracts were incubated with either an HA antibody or mouse IgG control. Presence of c-Raf in immunoprecipitates was determined using a c-Raf antibody and RanBPM expression was verified using HA, compared to 5% input extract. (B) RanBPM shRNA Hela cells were transfected with GST-ΔN-c-Raf and either pCMV empty vector or RanBPM si-mt, or with RanBPM si-mt and GST empty vector, and whole cell extracts were prepared 48 h post-transfection. Activated c-Raf was pulled down using glutathione-agarose beads, the presence of RanBPM was assessed using an HA antibody, and c-Raf expression was determined using a GST antibody, compared to 5% input extract. (C) Co-imunoprecipitation of Hsp90 with c-Raf. Extracts from Hela control (C) and RanBPM shRNA cells (2–7) were immunoprecipitated with c-Raf or mouse IgG control antibodies. Equal amounts of immunoprecipitated c-Raf from control and RanBPM shRNA cells were run on SDS-PAGE and analyzed by western blot with Hsp90 and c-Raf antibodies. Inputs represent 5% of the total protein used for immunoprecipitation. (D) Hela RanBPM shRNA cells were transfected with empty vector (-) or RanBPM si-mt, and whole cell extracts prepared 48 post-transfection were immunoprecipitated and analyzed as in C.