HIF-1a Stabilization by Bcl-2 Figure 2. bcl-2 promotes HIF-1a protein stability preventing its ubiquitin-mediated degradation. (A) Western blot analysis (left panel) and quantification (right panel) of HIF-1a protein expression in M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia for the indicated time. (B) Pulse analysis of HIF-1a protein synthesis rate in cells exposed to [35S]–labeled methionine and cysteine for the indicated time. (C) Western blot analysis (left panel) and quantification (right panel) of HIF-1a protein expression in cells exposed to hypoxia for 24 h and then treated with Cyclohexamide (CHX, 50 mg/ml) for the indicated time. (D) Pulse-chase analysis of HIF-1a protein (left panel) and quantification (right panel) in cells plated under dense conditions, pulsed for 45 min with [35S]–labeled methionine and cysteine and chased for the indicated time. (B,D) Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1a antibody and subjected to SDS-PAGE. (E) Western blot analysis of HIF-1a ubiquitination in the cells exposed to MG132 (10 mM, 6 h) or to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1a antibody and then the Western blot analysis was performed using anti-Ubiquitin antibody. (A,C) b-actin protein amounts are used to check equal loading and transfer of proteins and to quantify relative HIF-1a protein levels. (A–E) Western blot, pulse and pulse-chase analyses representative of two independent experiments with similar results are shown. (A,C,D) Densitometric analysis (right panel) of the relative Western blot or Pulse-chase analysis (left panel) was performed using Molecular Analyst Software and normalized with relative controls. doi:10.1371/journal.pone.0011772.g002 transfectants, but not in the control cells. Next, we tested by immunoprecipitation experiments whether the effect of bcl-2 on HIF-1a stabilization is due to an impairment of HIF-1a ubiquitination. As shown in Figure 2E, higher levels of ubiquitinated HIF-1a were found in control cells either treated with the proteasome inhibitor MG132 under normoxia, either exposed to hypoxia, when compared to levels of ubiquitinated HIF- 1a found in bcl-2 transfectants exposed to the same conditions. Taken together, all these data demonstrate that under hypoxia bcl-2 overexpression modulates HIF-1a expression at a post-translational level through the stabilization of the HIF-1a protein. bcl-2 protein interacts with HIF-1a protein To test whether the effect of bcl-2 on the stability of HIF-1a is due to their functional cooperation, we tested the eventual interaction between bcl-2 and HIF-1a protein by immunoprecip- itation experiments. When immunoprecipiatation was carried out using an antibody against bcl-2 protein and Western blot analysis was performed using antibodies that specifically recognizing HIF- 1a protein, bcl-2 was found to be immunoprecipitated with HIF- 1a protein in control cells and bcl-2 overexpressing clones after exposure to hypoxia, even though the bcl-2/HIF-1a immuno- complex was more evident in bcl-2 transfectants when compared to control cells (Figure 3A). To confirm the interaction between endogenous HIF-1a and bcl-2, the cells were treated with MG132 to accumulate similar levels of HIF-1a protein in all the cells, then immunoprecipitation experiments were performed using an anti- HIF-1a antibody and the bcl-2/HIF-1a immunocomplex were analyzed by Western blot using an anti-bcl-2 antibody (Figure 3B). Under these conditions, in spite of similar levels of PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11772