JCS03657 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Adobe PDF

• Supplemental Table S2 -

  Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. P-Y416 can be used as a marker of Src activation. (Left) Lysates from untransfected SYF^−/− were compared with cells expressing Src-WT. Activated Src was detected using an anti-P-Y416-SFK antibody (upper panel) with anti-actin as a loading control (lower panel). (Right) SYF^−/− cells stained with this anti-P-Y416-SFK antibody show no background staining. Bars, 25 μm.

• Supplemental Figure S2 -

  Fig. S2. Localization and activation of endogenous SFKs is similar to that of over-expressed proteins. (A) Wild-type MEFs were maintained in serum free medium (upper panels) or stimulated with PDGF (lower panels). SFK was detected using anti-Src, anti-Yes or anti-Fyn antibody (FITC secondary). Solid arrows in higher magnification images of boxed regions show SFK at the cell periphery and broken arrows indicate SFKs at the perinuclear region of the cell. Bars, 25 μm. (B) Endogenous Src (left panels), Yes (middle panels) or Fyn (right panels) were immunoprecipitated from unstimulated or PDGF-stimulated (25 ng/ml for 30 minutes) wild-type MEFs then an anti-P-Y416-SFK antibody was used to detect active protein (lower panels).

• Supplemental Figure S3 -

  Fig. S3. Src, Yes and Fyn can be detected at the tips of actin clouds. SYF^−/− cells were treated with cytochalasin D for 1 hour then washed twice in DMEM plus 10% FBS for 15 minutes to synchronize initiation of Src translocation and actin re-polymerization. Total protein was visualized using an anti-Src, anti-Yes or anti-Fyn antibody (FITC secondary), and actin with TRITC-phalloidin. Dotted arrows indicate actin clouds. Higher magnification of the boxed regions are shown in the insets. Bars, 25 μm.

• Supplemental Figure S4 -

  Fig. S4. Src is detected in both cytoplasmic and membrane fractions. SYF^−/− cells expressing Src-WT-GFP were treated with 10 μM L744832 (a farnesyl transferase inhibitor; FTI) for 2 hours prior to stimulation with PDGF. Lysates were homogenized and centrifuged for 1 hour at 100,000 g to give a cytosolic fraction (C) and a membrane fraction (M). Samples were analyzed by western blot using an anti-Src antibody.