Fig. 4.

Src–Eps8 complexes localize to autophagosomes in the absence of FAK. Eps8, Src or Src pY416 were immunoprecipitated (IP) from FAK WT or FAK^−/− cell lysates using (A) anti-Eps8, (B) anti-Src or (C) anti-Src pY416 antibodies followed by western blotting analysis with antibodies as indicated (lower panels). Anti-β-actin antibody was used as a loading control. (D) FAK WT and FAK^−/− cells were grown on glass coverslips, fixed and stained using anti-Src pY416 and anti-Eps8 (left hand panels) or anti-LC3B and anti-Eps8 (right hand panels). Solid arrows indicate focal adhesions (upper panels) and dashed arrows indicate internalized active Src or Eps8 (lower panels). Scale bars: 20 µm. Insets show a magnified view of the boxed area. A quantification is shown below the images. Results are mean±s.d. *P<0.001 (Student's t-test). (E) SCC subclone 1-1 and subclone 1-2 cells were suspended in PBS for 1 h and cytospins were prepared and stained for Src pY416 and DAPI. Solid arrows indicate focal adhesions (upper panels) and dashed arrows indicate internalized active Src or Eps8 (lower panels). Results are mean±s.d. The quantifications are representative of 100 cells from three independent experiments. Scale bars: 20 µm.