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Fig. 5 | Molecular Medicine

Fig. 5

From: Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Regulates the Signaling Mechanism of the Rapid Nongenomic Action of Dexamethasone on Actin Cytoskeleton

Fig. 5

Tyrosine phosphorylation (A) and expression (B) of FAK (I) and paxillin (II) following treatment of Ishikawa cells with cytochalasin B (CB) and DEX. (IA) Ishikawa cells were serum starved overnight and then treated with CB (1.2µM) for 2 hr. Subsequently, DEX was introduced at a final concentration of 10−7 M for 15 min. Cell lysates were prepared as described in Materials and Methods. Proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK monoclonal antibody. Heavy and light chains of the anti-phosphotyrosine IgG used to immunoprecipitate proteins phosphorylated on tyrosine residues are also shown. IP, immunoprecipitation; WB, Western blot. (IB) Experiment was performed as described in IA. Cell lysates were immunoprecipitated with anti-FAK monoclonal antibody and after separation of immunoprecipitates on SDS-polyacrylamide gradient (4–20%) gel, and proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK monoclonal antibody. (IIA) Ishikawa cells were serum starved overnight and then treated with CB (1.2 µM) for 2 hr. Subsequently, DEX was introduced at a final concentration of 10−7 M for 15 min. Cell lysates were prepared as described in Materials and Methods. Proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin monoclonal antibody. Heavy and light chains of the anti-phosphotyrosine IgG used to immunoprecipitate proteins phosphorylated on tyrosine residues are also shown. IP, immunoprecipitation; WB, Western blot. (IIB) Experiment was performed as described in IA. Cell lysates were run on SDS-polyacrylamide gel (12%), and proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin monoclonal antibody.

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