Inhibition of PI3K activity contributes to AITC-induced activation of PP1/PP2A and mitochondrial translocation of cofilin. (A) U937 cells were treated without or with various concentrations of AITC for 24 h, or treated with 20 μM AITC for different time intervals as indicated. Total cellular extracts were prepared and subjected to Western blot analysis using antibodies against phospho-PI3K (p-PI3K), PI3K, phospho-Akt [p-Akt (Ser473)], and Akt. Cells were pretreated with 20 μM LY294002, a specific PI3K inhibitor, for 2 h, followed by treating with 5 μM AITC for 24 h. (B) Total cellular extracts, cytosolic and mitochondrial fractions were prepared and subjected to Western blot analysis. (C) Cell lysates were prepared and subjected to immunoprecipitation using anti-PI3K antibody. The associated PP1 and PP2A were determined using immunoblotting. (D) Apoptosis was determined by flow cytometry. Error bars represent means ± SD (n=3). **P < 0.01. (E) Total cellular extracts and cytosolic fractions were prepared and subjected to Western blot analysis using antibodies as indicated.