Figure 2. Ambra1 interacts with Src and mediates trafficking of active Src to autophagosomes.
(A, B) Src (A) or pSrc Y416 (B) were immunoprecipitated from FAK-WT and FAK -/- cell lysates using anti-Src agarose or anti-pSrc Y416 antibody, followed by western blot analysis with anti-Ambra1, anti-pSrc Y416 and anti-Src. Relative ratios of Ambra1/Src and Ambra1/pSrc interactions were calculated by densitometry. (C) FAK-WT and FAK -/- cells were seeded onto glass coverslips, fixed and stained using anti-pSrc Y416, anti-Ambra1 and DAPI. Scale bars, 20 μm. (D) SCC FAK-WT and FAK -/- cells were grown on glass coverslips, fixed and stained with anti-Ambra1, anti-LC3B and DAPI. Scale bars, 20 μm. (E) LC3B was immunoprecipitated from SCC FAK-WT and FAK -/- cell lysates using anti-LC3B, followed by western blot analysis with anti-Ambra1 and anti-LC3B. Relative ratios of LC3B II/LC3B I as well as the Ambra1/LC3B and Ambra1/LC3B II interactions were calculated by densitometry. (F–J) SCC FAK-WT and FAK -/- cells were transiently transfected with either a pool (F–H) or two individual siAmbra1 siRNAs (F, I, J). The cells were grown on glass coverslips, fixed and stained with anti-pSrc Y416, anti-Paxillin and DAPI. (G, I) Representative immunofluorescence images. Scale bars, 20 μm. (H, J) Quantification of internalised active Src. n = 3. Error bars, s.d. p<0.001. Colocalisation (Costes r value from five cells) was analysed using the ImageJ plugin JaCoP.
DOI: http://dx.doi.org/10.7554/eLife.23172.007
Figure 2—figure supplement 1. Ambra1 interacts with Src and mediates pSrc trafficking.
