Fig. 2. Activation of all three SFKs requires an intact actin cytoskeleton. (A) SYF^–/– cells expressing Src-WT, Yes-WT or Fyn-WT were treated with cytochalasin D (0.3 µg/ml) for 1 hour prior to PDGF stimulation. Localization of active SFK was detected with an anti-P-Y416-SFK antibody (Cy5 secondary), total protein with an anti-Src, anti-Yes or anti-Fyn antibody (FITC secondary) and actin with TRITC-phalloidin. Bars, 25 µm. (B) Lysates from SYF^–/– cells expressing Src-WT, Yes-WT or Fyn-WT maintained in suspension (Sus) or plated on fibronectin (FN) for 1 hour (10 µg/ml) were immunoblotted using anti-Src, anti-Yes, anti-Fyn (upper panels) and anti-P-Y416-SFK antibodies (lower panels). Quantification shown in Table S1 in supplementary material.