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Fig. 4. Palmitoylation contributes to RhoB-dependence of intracellular targeting. (A) SYF^–/– cells expressing Src-GFP S3C/S6C, Yes C3S or Fyn-GFP C3S/C6S were maintained in serum-free medium (upper panels) or stimulated with PDGF (lower panels). Solid arrows indicate localization of active SFK at cell periphery whereas broken arrows indicate inactive protein at the perinuclear region. (B) SYF^–/– cells expressing Src-GFP S3C/S6C or Fyn-GFP C3S/C6S with Myc-RhoB or Myc-RhoD were stimulated with PDGF. Solid arrows in higher magnification images indicate colocalization between active SFK and Myc-Rho. (C) RhoB^+/+ (upper panels) or RhoB^–/– cells (lower panels) expressing Src-GFP S3C/S6C, Yes C3S or Fyn-GFP C3S/C6S were plated on fibronectin for 1 hour. Solid arrows indicate active SFK at the cell periphery and broken arrows inactive SFK at the perinuclear region. Localization of active SFK was detected using an anti-P-Y416-SFK antibody (Texas Red or Cy5 secondary), total protein using an anti-Yes antibody (FITC secondary) and Myc-Rho was visualized using an anti-Myc antibody (Texas Red secondary). Bars, 25 µm. Quantification shown in the Tables S1 and S2 in supplementary material.

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