Fig. 4.

Signalling proteins localise to Src-containing autophagosomes. FAK −/− cells were fixed and stained with (A) anti-p-PDK1 S241 (upper panels), anti-p-Akt S473 (lower panels) or (B) anti-p-p70S6K T389 (upper panels), anti-mTOR (lower panels) and with anti-p-Src Y416 and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate co-localisation while broken arrows show its absence. Scale bars: 20 μm. (C) Amount of co-localisation per cell was calculated by measuring by the distance between the intensity peaks of the different fluorescent signals. Results are presented as mean ± s.d. (n = 20 intensity peaks from 5 cells). (D) Src was immunoprecipitated from SCC cells and immunoblotting performed with anti-p70S6K and anti-Src. (E) Model of signalling at Src positive autophagosomes. Schematic depicting how the use of inhibitors influences the trafficking of active Src to autophagic puncta in the absence of FAK.