Figure 3.
Regulation of the cellular phosphorylation state of cofilin and its impact on the PLD response. HEK-293 cells were stimulated without (−) or with (+) 1 mM carbachol for the indicated periods of time (A), or transfected with empty vector (Control), c-myc-tagged wild-type slingshot 1L, inactive slingshot 1L (C393S slingshot 1L) or VSV-G-tagged 14-3-3ζ, and stimulated with carbachol for 15 s (B). Phosphorylation of cofilin (P-Cofilin) and the total cellular content of cofilin were detected in cell lysates with anti-phospho-cofilin and anti-cofilin antibodies, respectively. Data are representative of three experiments. (C) Stimulated [3H]PtdEtOH accumulation was determined in the presence of 1 mM carbachol (Carb) or 100 ng/ml EGF. Data shown are means±s.e. (n=3–4). The immunoblots show expression of c-myc-tagged slingshot 1L and myc-tagged inactive slingshot 1L (C393S slingshot 1L), or VSV-G-tagged 14-3-3ζ. *P<0.05.