Fig. 4

Tyrosine phosphorylation (A) and expression (B) of FAK (I) and paxillin (II) in Ishikawa cells treated with DEX and pervanadate. (IA) Ishikawa cells were serum starved overnight and then exposed for 30 min to 1 mM sodium orthovanadate in conjunction with 3 mM hydrogen peroxide [pervanadate (PER), lane 3]. Subsequently, DEX was introduced at a final concentration of 10⁻⁷ M for 15 min (lane 4). 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, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK monoclonal antibody. (ILA) Ishikawa cells were serum starved overnight and then exposed for 30 min to 1 mM sodium orthovanadate in conjunction with 3 mM hydrogen peroxide [pervanadate (PER), lane 3]. Subsequently, DEX was introduced at a final concentration of 10⁻⁷ M for 15 min (lane 4). 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 IIA. Cell lysates were run on SDS-polyacrylamide gel (12%), and proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin monoclonal antibody.