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. 2017 Mar 31;6:e23172. doi: 10.7554/eLife.23172

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. (FJ) SCC FAK-WT and FAK -/- cells were transiently transfected with either a pool (FH) 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—source data 1. COSTES r values for immunofluorescence images and percentage of cells with internalised pSrc.
COSTES mean and s.d. values for Figures 2C and D are shown. Mean percentage and s.d. values of cells with internalised pSrc upon transient Ambra1 knockdown by siRNA in SCC FAK-WT and -/- cells are shown (Figures 2H,J).
DOI: 10.7554/eLife.23172.008

Figure 2.

Figure 2—figure supplement 1. Ambra1 interacts with Src and mediates pSrc trafficking.

Figure 2—figure supplement 1.

(A) Nuclei from SCC FAK-WT and FAK -/- cells were isolated using sucrose gradient centrifugation. Lysates were immunoblotted for Ambra1, GAPDH (cytosolic marker) and Lamin A/C (nuclear marker). (B) Relative ratio of Ambra1/Lamin A/C was calculated by densitometry. WCL, whole cell lysate. Error bars, s.d. (C) Colocalisation (Costes r value from five cells) of pSrc Y416/Paxillin upon Ambra1 knockdown by siRNAs was analysed using the ImageJ plugin JaCoP. Error bars, s.d. p<0.01 (*) and p<0.05 (#). (D) FAK-WT and FAK -/- cells were transiently transfected with either a pool or two independent Ambra1 siRNAs. Cell lysates were subjected to western blot analysis using anti-Ambra1, anti-pSrc Y416 and anti-Src. Anti-GAPDH was used as a loading control. (E) The relative ratios of pSrc/GAPDH were calculated using densitometry. n = 3. Error bars, s.d. p<0.01 (*) and p<0.05 (#).