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

Figure 3.

Figure 3

Src phosphorylates mATG9 at Y8 to promote constitutive trafficking of mATG9. (A) HeLa cells were transfected with Src-GFP or kinase-dead (KD) Src for 24 h, and collected for immunoprecipitation with anti-mATG9 antibody. (B) HeLa cells were treated with the Src kinase inhibitors PP2 (10 μM) or SU6656 (10 μM) for 12 h and collected for immunoprecipitation with anti-mATG9 antibody. (C, D) HEK293T cells were co-transfected with mATG9-Myc, 3×Flag-AP1/2M1 and GFP vector, Src-GFP or KD Src for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (E, F) HEK293T cells were co-transfected with WT mATG9 or the Y8F mutant, 3×Flag-AP1/2M1 and Src-GFP for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (G) HeLa cells were co-transfected with WT mATG9-Myc or the Y8F mutant and 3×Flag-AP1/2M1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (H, I) HeLa cells were co-transfected with mATG9 and 3×Flag-AP1/2M1 for 24 h, and then treated with vehicle or the Src kinase inhibitors PP2 (10 μM) or SU6656 (10 μM) for 12 h. Cells were collected for immunoprecipitation with anti-Flag antibody. (J) U2OS cells were co-transfected with WT mATG9-Myc or the indicated mutants (red) and GFP vector, Src-GFP or KD Src (green) for 24 h, and then fixed and immunostained with anti-Myc antibody. Scale bar, 10 μm. See also Supplementary information, Figure S3B-S3C.