Fig. 6.

SNX9 is phosphorylated by Src. (A) NIH-3T3 or NIH-Src were treated with Src inhibitor SU6656 or DMSO (control). Cells were then stained for F-actin and for endogenous SNX9. Scale bars: 10 μm. (B) Western blot comparing exogenous SNX9 phosphorylation in NIH-Src cells transiently expressing GFP–SNX9, mock-treated or treated with SU6656. Tyrosine phosphorylation was evaluated on immunoprecipitated GFP–SNX9 using an anti-phospho-tyrosine antibody. GAPDH was used as loading control. (C) Schematic presentation of SNX9 protein domains. Tyrosines, identified by mass spectrometry (see Fig. S4B), phosphorylated by Src are marked in red. (D) HA–Src and V5–WT-SNX9 or the indicated mutants were transiently co-expressed in HEK293 cells. V5–SNX9 was then immunoprecipitated and tyrosine phosphorylation was assessed using an anti-phospho-tyrosine antibody. 5YF represents the quintuple mutant, where all phosphorylated tyrosines were mutated (Y177F, Y239F, Y269F, Y294F and Y561F).