Figure 3

AITC induces dephosphorylation of cofilin by PP1 and PP2A. (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 PP1, PP2A and slingshot (SSH). Cells were pretreated with 50 nM calyculin, a PP1/PP2A inhibitor, for 2 h, followed by treating with 20 μM AITC for 24 h. (B) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies against PP1, PP2A, p-cofilin, and cofilin. (C) Cell lysates were prepared and subjected to immunoprecipitation using anti-cofilin antibody. The associated PP1, PP2A, SSH, and cofilin were determined using immunoblotting. (D) Cell apoptosis was determined by Annexin V/PI staining and 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.