Figure 6.
Interaction between endogenous and spatially restricted Bcl-2 and Beclin-1 in starvation and after addition of ABT737. (A) Immunoprecipitation of endogenous Bcl-2 and Beclin-1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient-free EBSS or with ABT737 (1 μM). (B) Immunoprecipitation of wild-type and ER-targeted Bcl-2 with Beclin-1. Cells stably transfected with wild type, ER- and mitochondrion-targeted Bcl-2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl-2 and immunodetection of Beclin-1. (C, D) Immunoprecipitation of Bcl-2 and Beclin-1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl-2 and immunodetection of Beclin-1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations.