2ME2 induces mitochondrial release of cytochrome-c and Smac.

(A,B) MM.1S cells were treated with 2ME2 (3 μM) or Dex (0.05 μM) and were harvested at 48 hours. Cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting with anti–cyto-c (A, upper panel) or anti-Smac (B, upper panel) antibodies. As a control for equal loading of proteins, filters were also reprobed with anti-SHP2 antibody (A and B, lower panels). Blots are representative of 3 independent experiments. (C,D) Dox-40 and LR-5 MM cells were treated with 2ME2 (3 μM) and harvested at 48 hours. Cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting with anti–cyto-c (C, upper panels) or anti-Smac (D, upper panel) antibodies. As a control for equal loading of proteins, filters were reprobed with anti-SHP2 antibody (C and D, lower panels). Blots are representative of 3 independent experiments. Densitometric analysis of the immunoblot demonstrated that 2ME2 induced a 4- to 5-fold increase in the cytosolic cyto-c and Smac levels compared with untreated cells.