American Society of Hematology

Figure 7.

$Figure 7. TGF-β1 induces phosphorylation of RhoA facilitating RhoA-RhoGDI complex formation. (A) Cells were treated with 100 μM forskolin or 5 ng/mL TGF-β1 for the indicated times and lysed. Phosphorylated RhoA was immunoprecipitated with anti–phospho-Ser antibody and protein-A–conjugated agarose and detected by Western blotting with anti-RhoA antibody. (B) Cells were treated with 100 μM forskolin or 5 ng/mL TGF-β1 in the presence or absence of 30 μM H89 for 30 minutes and lysed. Immunoprecipitation and immunoblotting were performed as in panels A and B. (C) Cells were preincubated with 30 μM H89 for 30 minutes and then treated with TGF-β1 for 12 hours or 24 hours. GTP-RhoA in cell lysates was bound to GST-RBD and GSH-Sepharose beads and revealed by Western blot using anti-RhoA antibody. (D) Cells were preincubated with 30 μM H89 for 30 minutes, then with TGF-β1 for 1 hour or 24 hours, and lysed. RhoGDI antibody was added to the cell lysates, and immunocomplexes were precipitated with Protein-A–conjugated agarose. Proteins were then fractionated using 2 × Laemmli buffer and analyzed by Western blotting with anti-RhoA and anti-RhoGDI antibodies.$

TGF-β1 induces phosphorylation of RhoA facilitating RhoA-RhoGDI complex formation. (A) Cells were treated with 100 μM forskolin or 5 ng/mL TGF-β1 for the indicated times and lysed. Phosphorylated RhoA was immunoprecipitated with anti–phospho-Ser antibody and protein-A–conjugated agarose and detected by Western blotting with anti-RhoA antibody. (B) Cells were treated with 100 μM forskolin or 5 ng/mL TGF-β1 in the presence or absence of 30 μM H89 for 30 minutes and lysed. Immunoprecipitation and immunoblotting were performed as in panels A and B. (C) Cells were preincubated with 30 μM H89 for 30 minutes and then treated with TGF-β1 for 12 hours or 24 hours. GTP-RhoA in cell lysates was bound to GST-RBD and GSH-Sepharose beads and revealed by Western blot using anti-RhoA antibody. (D) Cells were preincubated with 30 μM H89 for 30 minutes, then with TGF-β1 for 1 hour or 24 hours, and lysed. RhoGDI antibody was added to the cell lysates, and immunocomplexes were precipitated with Protein-A–conjugated agarose. Proteins were then fractionated using 2 × Laemmli buffer and analyzed by Western blotting with anti-RhoA and anti-RhoGDI antibodies.

This Feature Is Available To Subscribers Only