Skip to main content
. 2009 Jun 1;20(11):2650–2660. doi: 10.1091/mbc.E09-02-0131

Figure 4.

Figure 4.

AngII dephosphorylates cofilin through the ROS-dependent dissociation of SSH-1L from 14-3-3. (A–F) HeLa cells were cotransfected with AT1R and Nox1s (Nox1, NoxO1, and NoxA1) for 18 h and then serum-starved for 16 h. (A) The cells were stimulated with 0.1 μg/ml AngII for the indicated times. Cell lysates were immunoblotted with antibodies to phosphorylated cofilin (p-Cofilin) or cofilin (t-Cofilin). (B) The cells were pretreated with or without 100 nM wortmannin (Wort) or 10 μM DPI for 30 min and then stimulated with 0.1 μg/ml AngII for the indicated times. Cell lysates were immunoblotted with antibodies to phosphorylated cofilin (p-Cofilin) or cofilin (t-Cofilin). (C) The cells cotransfected with or without HA-14-3-3ζ-wt or HA-K49E mutant were stimulated with 0.1 μg/ml AngII for the indicated times. Cell lysates were immunoblotted with antibodies to phosphorylated cofilin (p-Cofilin), cofilin (t-Cofilin), or HA (HA-14-3-3ζ). (D) The cells cotransfected with or without GST-SSH-1L or HA-14-3-3ζ-wt were pretreated with or without 10 μM DPI or 100 nM wortmannin (Wort) for 30 min and then stimulated with 0.1 μg/ml AngII for the indicated times. GST pulldown of proteins from cell lysates and the total cell lysates (TCL) were immunoblotted with antibodies to GST (GST-SSH-1L) or HA (HA-14-3-3ζ). (E) Cells were stimulated with 0.1 μg/ml AngII for the indicated times. Cell lysates were prepared and proteins labeled with 5′-iodoacetamide fluorescein, as in Materials and Methods. HA-14-3-3ζ was immunoprecipitated with anti-HA antibodies, followed by immunoblotting with anti-FITC antibodies. The total immunoprecipitated HA-14-3-3ζ was analyzed by immunoblotting with anti-HA antibodies. Loss of anti-FITC reactivity is indicative of protein oxidation. (F) The cells were pretreated with or without 10 μM DPI or 100 nM wortmannin (Wort) for 30 min and then stimulated with 0.1 μg/ml AngII for 30 min. Cell lysates were prepared and proteins were labeled with 5′-iodoacetamide fluorescein. The HA-14-3-3ζ was immunoprecipitated with anti-HA antibodies, followed by immunoblotting with anti-FITC antibodies. The total immunoprecipitated HA-14-3-3ζ was analyzed by immunoblotting with anti-HA antibodies. (A–F) Representative experiments from three separate experiments are shown.