Fig. 6. Slingshot is the major regulator of actin-dependent cofilin phosphorylation. (A) Ssh RNAi blocks jasplakinolide-induced loss of cofilin phosphorylation. Control S2R⁺ cells and cells treated with dsRNA targeting Ssh were treated with jasplakinolide (1 µg/ml) for the times indicated and P-cofilin and -actin levels assessed in the lysates by immunoblotting. (B) LIMK RNAi does not reverse Ssh dsRNA blockade of jasplakinolide-induced loss of cofilin phosphorylation. Cells treated with dsRNA targeting Ssh (SSH) or Ssh and LIMK together (SSH/LIMK) were treated with jasplakinolide (1 µg/ml) for the indicated times, and P-cofilin and -actin levels assessed in lysates by immunoblotting. (C) The effect of LIMK and Ssh on latrunculin B-induced cofilin-phosphorylation. Cells treated with control dsRNA and dsRNA targeting LIMK or LIMK and SSH together (LIMK/SSH) were treated with latrunculin B (1 µg/ml) for the indicated times, and P-cofilin and -actin levels in lysates were determined by immunoblotting. (D) Loss of actin expression induces cofilin phosphorylation. P-cofilin and -actin levels were determined by immunoblotting in lysates of control cells (untreated) and cells treated with dsRNAs targeting actin, cofilin and LacZ.