HIF-1a Stabilization by Bcl-2 Figure 4. bcl-2 interacts with HIF-1a in the nucleus. (A) Western blot analysis of bcl-2 and HIF-1a protein expression in nuclear (Nucl) and cytoplasmic (Cyto) protein extracts of M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5) clones exposed to hypoxia or to normoxia for 24 h. LaminA/C (Lam A/C) and b-tubulin were used as markers for nuclear and cytoplasmic fraction, respectively. b-actin protein amounts are used to check equal loading and transfer of proteins. (B) Confocal laser scanning microscopy of immunofluorescence staining performed on Bcl2/5 stably overexpressing clone exposed to hypoxia or to normoxia for 24 h. Fixed cells were labelled with anti-bcl-2 (green) or anti-HIF-1a (red) antibodies. Nuclei were visualized using TO-PRO3H staining (blue). (C) Analysis of HIF-1a/bcl-2 interaction in Bcl2/5 stably overexpressing clone exposed to hypoxia for 24 h. Nuclear (Nucl) and cytoplasmic (Cyto) protein extracts were immunoprecipitated (IP) with anti-HIF-1a or anti-bcl-2, respectively, or control antibody (IgG) and then the Western blot analysis was performed using anti-bcl-2 or anti-HIF-1a antibodies. (A–C) Western blot and confocal analyses representative of two independent experiments with similar results are shown. doi:10.1371/journal.pone.0011772.g004 degradation of HIF-1a protein, we generated M14 cell line stably expressing wild type form of HIF-1a (HIF-1a wt) or hydroxylation- resistant (P402A/P564A) form of HIF-1a (HIF-1a PP/AA). These cells were then transiently transfected with an empty vector or with a vector encoding bcl-2 protein and HIF-1a expression and transcriptional activity were analyzed under hypoxic conditions. As depicted in Figure 5, bcl-2 overexpression significantly increased the levels of both exogenous wt and hydroxylation- resistant form of HIF-1a (Figure 5A) and it also enhanced HRE- dependent transcriptional activity (Figure 5B). As expected, PHD2 overexpression inhibited the expression of HIF-1a wt and HRE-dependent transcriptional activity while it did not abrogate the expression and activation of reporter gene transcription in cells expressing HIF-1a protein containing the proline-to-alanine substitutions (Figure 5B). The discovery that bcl-2 had similar effects on the wt and mutant form of HIF-1a indicated that bcl-2 PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11772