Figure 3. IFITM3 is in the centre of an Ambra1, FAK and pSrc trafficking network.

(A) Network analysis of Ambra1-, FAK- and pSrc Y416-interacting proteins that are involved in trafficking processes. Solid lines indicate protein-protein interactions identified in the mass spectrometry datasets used for the interaction map. Dotted lines indicate Ambra1–FAK/pSrc interactions, which have been previously identified and verified by immunoprecipitation. (B) Ambra1 was immunoprecipitated from FAK-WT and FAK -/- cell lysates using anti-Ambra1 antibody, followed by western blot analysis with anti-Dctn1 and anti-Ambra1. (C–E) Ambra1 (C), FAK (D) or pSrc Y416 (E) were immunoprecipitated from FAK-WT and -/- cell lysates, followed by western blot analysis with anti-IFITM3, anti-Ambra1, anti-FAK and anti-pSrc Y416. Anti-GAPDH served as a loading control. Relative ratios of Dctn1/Ambra1, IFITM3/Ambra1, IFITM3 and IFITM3/pSrc interactions were calculated by densitometry.

DOI: http://dx.doi.org/10.7554/eLife.23172.010

Figure 3—figure supplement 1. IFITM3 influences the Src-FAK complex.

(A) Focal adhesions from SCC FAK-WT and FAK -/- cells were crosslinked and isolated for western blot analysis with the indicated antibodies. Paxillin served as a marker for focal adhesions, Lamin A/C was used as a nuclear marker and GAPDH represented a cytosolic marker. WCL, whole cell lysate; FA, focal adhesions. SCC FAK-WT and -/- cells were transiently transfected with siIFITM3 and pSrc Y416 (B) and FAK (D) were immunoprecipitated from FAK-WT and FAK -/- cell lysates using anti-pSrc Y416 and anti-FAK 4.47 agarose, followed by western blot analysis with anti-Ambra1, anti-FAK, anti-IFITM3, anti-pSrc Y416 and anti-Src. Anti-GAPDH served as a loading control. (C, E) Relative ratios of Ambra1/pSrc (C) as well as pSrc/FAK and Src/FAK (E) interactions were calculated by densitometry. n = 3. Error bars, s.d. p<0.05 (#).

DOI: http://dx.doi.org/10.7554/eLife.23172.011