Fig. 5. PI3K-induced changes in cofilin phosphorylation are the result of actin cytoskeletal remodelling. (A) LIMK and Ssh are regulators of cofilin phosphorylation. S2R⁺ cells were left untreated (Ctrl) or were treated with dsRNA for knockdown of expression of LIMK, Ssh and cofilin for 5 days. Cells were lysed and P-cofilin and -actin levels assessed in the lysates by immunoblotting. (B) Immunoblotting of P-cofilin, P-Akt and -actin in lysates of dsRNA-treated cells stimulated with insulin. S2R⁺ cells were cultured in the presence of dsRNA targeting LacZ (control), Rac1+2 and Ssh and then treated with 10 µg/ml insulin for the indicated times prior to lysis and immunoblotting. (C) P-cofilin and -actin levels were assessed in extracts from S2R⁺ cells treated with jasplakinolide (1 µg/ml), latrunculin B (1 µg/ml) or cytochalasin D (2 µg/ml) for the times indicated. (D) P-cofilin, P-Akt and -actin levels are shown for S2R⁺ cells pre-treated for 10 minutes with latrunculin B (1 µg/ml) or jasplakinolide (1 µg/ml) followed by 20 minutes of insulin treatment (10 µg/ml) or vehicle alone.