HIF-1a Stabilization by Bcl-2 Figure 6. bcl-2 forms a complex with HSP90 and HIF-1a proteins. (A) Western blot analysis of HIF-1a protein expression in M14 control cells (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones treated with 17-AAG under hypoxia or exposed to normoxia for 24 h. (B) HRE- dependent transcriptional activity in the cells treated with 17-AAG from 0.05 to 2 mM under hypoxia or exposed to normoxia for 24 h. Relative luciferase activity of each sample was normalized to untreated cells exposed to normoxic conditions. Results represent the average 6 SD of 3 independent experiments performed in triplicate. p values were calculated relative to untreated cells exposed to hypoxic conditions, *p#0.01. (C) Western blot analysis of HSP90 protein expression in parental M14 cells, control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones. (D) Analysis of HIF-1a/HSP90 interaction in the cells exposed to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1a or control (IgG) antibodies and then the Western blot analysis was performed using anti-HSP90 and anti-HIF-1a antibodies. (E) Analysis of HSP90/HIF-1a and HSP90/bcl-2 interactions in the cells exposed to hypoxia for 24 h. Cell lysates were immunoprecipitated (IP) with anti-HSP90 or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1a, anti-bcl-2 and anti-HSP90 antibodies. (F) Analysis of HIF-1a/HSP90 and HIF-1a/bcl-2 interactions in the cells treated with 0.5 mM 17-AAG for 24 h under hypoxia. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1a antibody and then the Western blot analysis was performed using specific anti-HSP90 and bcl-2 antibodies. (G) Analysis of HSP90/HIF-1a/ bcl-2 protein complex in the cells exposed to hypoxia for 24 h. Whole cell lysates were sequentially immunoprecipitated with anti-HIF-1a (IP1) and anti-bcl-2 antibodies (IP2) and then the Western blot analysis was performed using anti-HSP90 antibody. (A,C) b-actin protein amounts are used to check equal loading and transfer of proteins. doi:10.1371/journal.pone.0011772.g006 HIF-1a/bcl-2 tri-complex. Overall these findings suggested that bcl-2 may promote stabilization of HIF-1a by increasing its ability to interact with the HSP90 chaperone complex, probably affecting its folding and maturation. HSP90b isoform is the mediator of HIF-1a induction by bcl-2 under hypoxic conditions The molecular chaperones HSP90 comprise two homologous proteins, HSP90a and HSP90b, that are encoded by distinct genes PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11772