Evaluation of Beclin-1 and Bcl-2 interaction.

A) EPCs were treated with CLC and RAD alone or in sequence and thereafter proteins were extracted. Then we performed western blot assay for the expression of the total Bcl2 and Beclin-1 proteins and immunoprecipitation of Bcl-2 and Beclin-1 subsequently blotted with either anti-Beclin-1 or Bcl-2 antibodies, respectively, for the evaluation of Bcl-2/Beclin-1 and Beclin-1/Bcl-2 complex formation as described in “Material and Methods”. Untreated cells, CTR; CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. The figure is representative of three different experiments that always gave similar results. B) Representation of the Bcl2/Beclin-1 complexes expressed as the ratio between the relative intensities of the bands associated with the Bcl-2/Beclin-1 and Beclin-1/Bcl-2 complexes versus the bands associated with total Bcl-2 and Beclin-1, respectively. The intensities of the bands were expressed as arbitrary units when compared to those of the untreated cells. The values are the mean of three independent experiments. SDs, standard deviations.