Fig. 2. Immunoprecipitated Beclin complex from U-251 cells contains hVps34 but not Bcl-2 or Bcl-X_L. (A) Cell lysates were prepared from parallel cultures as described in the Materials and Methods and 10% of each lysate was saved for immunoblot analysis. The remainder of each lysate was immunoprecipitated with either IgG against Beclin or a `control' IgG against an unrelated protein (SPARC, Santa Cruz Biotechnology). Equal aliquots of the immune complexes eluted from protein A Sepharose beads were probed by immunoblot analysis using the antibodies indicated at the left of each panel. The band appearing just below Beclin in the upper panel is a nonspecific crossreacting protein. (B) Immunoprecipitation was performed as described in panel A, except that a rabbit antibody against Bcl-2 was used for the pull-down. The immunoprecipitated protein complexes were probed with the antibodies indicated at the left of each panel.