FIG. 4.

Tyrosine phosphorylation of Tyr527 and Tyr416 during ECM contact. NIH 3T3 cells (1 × 10⁶) were serum starved for 24 h before being detached and maintained in suspension for 30 min at 37°C with gentle agitation. Cells were either kept in suspension or seeded onto fibronectin-treated dishes for the indicated times. Twenty-five micrograms of total lysate in cRIPA buffer was separated by 10% SDS-PAGE, and immunoblotting (WB) was performed with anti-phospho-Src (Tyr416 or Tyr527) or with c-Src antibodies for normalization. Normalized values for the phosphorylation of each tyrosine are reported in the plot. S, suspended cells; A, adherent cells. The times (in min) of adherence are indicated.