FIG 8.

Grb2 SH2 and C-terminal SH3 domains are required for cell spreading and migration and PTPα-Grb2 association. (A) MEFs treated with either control (Ctl) or Grb2 siRNA duplex or cotransfected with Grb2 siRNA and Grb2-myc-WT or Grb2-myc mutants were kept in suspension for 1 h and replated on FN-coated dishes for 15 min. Cells were fixed and immunostained with phalloidin (red) and myc (green). Bar, 20 μm. (B) Cells were plated on FN-coated coverslips for 48 h. A wound was created (0 h), and the cells were allowed to migrate into the cleared area for 14 h prior to staining with 1 nM calcein AM in medium. Bar, 250 μm. (C) The distance migrated by cells in two independent experiments as in panel B was measured as described in Materials and Methods, and the average is shown in the graph. (D) MEFs were transfected with Grb2-myc-WT or mutants. Anti-myc immunoprecipitates from adherent cell lysates were probed for PTPα and myc. Lysates were immunoblotted as shown.