Decreased Bcl-2 expression contributes to TSC1-deficient mast cell death. (A) Bcl-2 family protein expression. Cell lysates from IL-3 sufficient or depleted cultures for 10 hours were analyzed by immunoblot with the indicated antibodies. (B) qRT-PCR to determine Bcl-2 mRNA expression. (C) Decreased mitochondrial potential in TSC1KO BMMCs. Analysis of ΔΨ by flow cytometry using DiOC6. (D-E) Rescue of TSC1KO BMMCs from death by expressing Bcl-2 but not CA-Akt. TSC1-deficient BMMCs were infected with retroviruses encoding GFP plus Bcl-2, Akt-DD, or Myr-Akt. GFP-positive populations were used to determine virus-infected cells. After 48 hours incubation in media without IL-3, cells were stained and analyzed. (D) Representative FACS plots. (E) Graphical presentation of annexin V–positive apoptotic cells. Data shown are representative of 3 experiments (*P < .05; ***P < .001).