Down-regulation of Akt activity by ANT2 shRNA in SK-BR3 cells. (A) After 24 hours of transfection with ANT2 shRNA in SK-BR3, cells extracts were prepared for western blotting with anti-phospho Akt and anti-Akt antibodies. 17-AAG was used as a positive control. (B) To investigate if ANT2 plays a role in the regulation of Akt activity (phophorylated Akt level) in SK-BR3, cells were transfected with scramble shRNA, ANT2 shRNA, pcDNA, or pcDNA-ANT2 and cultured for 24 hours, then cells extracts were prepared for western blotting with anti-phospho Akt and anti-Akt antibodies. PI3K inhibitor, LY294002 was used as a positive control to inhibit PI3K/Akt signaling pathway. (C) To evaluate the effect of ANT2 shRNA-mediated HSP90 suppression on the stability and phosphorylation of Akt, cells were transfected with ANT2 shRNA for 24 or 48 hours and the cellular extracts were prepared for immuno-precipitation with anti-HSP90 antibody and subjected to western blotting with anti-phospho-Akt and anti-Akt antibodies.