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. 2016 Dec 9;27(2):184–201. doi: 10.1038/cr.2016.146

Figure 4.

Figure 4

Stimulation of Src kinase activity by EGF promotes retrograde trafficking of mATG9. (A) HeLa cells were serum-starved for 24 h, and then stimulated with 50 ng/ml hEGF for the indicated times. Cells were collected for immunoprecipitation with anti-mATG9 antibody and phospho-mATG9 was assessed by immunoblotting with a specific antibody against Y8. (B) HeLa cells stably expressing mATG9-Myc were transfected with 3×Flag-AP1/2M1, serum-starved for 24 h, and then stimulated with hEGF for the indicated times. Cells were collected for immunoprecipitation with anti-Flag antibody. (C-E) Atg9a KO HeLa cells reconstituted with wild-type mATG9 (C) or the SS1/2 (D) or Y8F (E) mutants were starved of serum for 24 h, and then stimulated with hEGF for the indicated times. Cells were fixed and immunostained with anti-Myc (red) and anti-EGFR (green) antibodies. Nuclei are stained with DAPI (blue). Boxed areas in the left panels are enlarged in the right panels. Scale bar, 10 μm. See also Supplementary information, Figure S3D-S3F.