Figure 8.
Inhibition of the Bcl-xL/Bax complex formation by IPAS. (a) Inhibition of interaction between Bcl-xL and Bax by IPAS. HEK293T cells were transfected with indicated combinations of expression plasmids, incubated in the presence of Z-VAD-FMK and analyzed by immunoprecipitation with antibody against FLAG followed by immunoblotting with antibody against HA. Immunoprecipitated HA-Bax was normalized to total HA-Bax in the lysates in each lane and the result is shown below. (b) Western blot analysis of subcellular localization of endogenous Bax in PC12 cells. Cells were treated with CoCl2 for indicated times and separated into cytosolic and membrane fractions, followed by immunoblotting with indicated antibodies. Bcl-xL and GAPDH were controls for cytosol and mitochondrial localization, respectively. (c) Inhibition of dissociation of the Bcl-xL/Bax complex by IPAS/HIF-3α siRNA treatment. PC12 cells were transfected with GFP or IPAS/HIF-3α siRNA and incubated with or without CoCl2 in the presence of Z-VAD-FMK for 16 h, and cell lysates were analyzed by immunoprecipitation with antibody against Bcl-xL followed by immunoblotting with antibody against Bax. (d) Inhibition of IPAS-induced activation of caspase-3 by Bcl-2 family members. PC12 cells were transfected with pCerulean, pCerulean-IPAS, pCerulean-IPAS+pBOS-3MycBcl-xL and pCerulean-IPAS+pBOS-3MycBcl-2, respectively, stained with active-caspase-3 antibody and Hoechst 33342 and observed using a fluorescence microscope. A minimum of 100 transfected cells with or without active caspase-3 staining per sample was counted and the result is shown at right. Data shown in bar graphs are averages±S.D. of three independent experiments. *P<0.05 for indicated comparison. **P<0.01 for indicated comparison. (e) A model for the action of IPAS in apoptosis. IPAS is bound to Bcl-xL and some other pro-survival Bcl-2 members on mitochondria and blocks the interaction of Bcl-xL with Bax, thus activating the pro-apoptotic function of Bax, followed by release of cytochrome c and activation of caspase-3. IPAS functions as a dual function protein involved in transcription repression of HIF-1α and apoptosis