The tyrosine phosphorylation of cortactin is important for Nck1 localization and N-WASp activity at invadopodium precursors for Arp2/3-dependent barbed end formation. (A) Representative images of EGF-induced barbed ends at invadopodium precursors in ctrl vs. Nck1 vs. p34 KD cells. Bar, 10 μm. (B) Quantification (normalized to 0 min) of barbed ends formed in response to EGF at invadopodium precursors in ctrl vs. Nck1 vs. p34 KD cells. P values are compared with ctrl siRNA at the same time point. n = number of invadopodium precursors from two independent experiments: ctrl 0 (264), 1 (287), 2 (275), Nck1 0 (223), 1 (356), 2 (375), p34 0 (130), 1 (201), 2 (259). (C) Representative images of GFP-Nck1 and cortactin in WT and 3YF cortactin cell lines (arrowheads show invadopodium precursors). Bar, 10 μm. (D) Quantification of fold enrichment of GFP-Nck1 at invadopodium precursors in WT and 3YF cell lines. n = number of invadopodium precursors: WT (29), 3YF (34). (E) Representative Western blot showing the coIP of endogenous cortactin and pY421-cortactin with endogenous Nck1. n = 3 independent experiments. (F) Representative images of N-WASp activity measured using the CFP/YFP FRET ratio in live cells expressing cortactin WT or 3YF (arrowheads show invadopodium precursors). Bar, 10 μm. (G) Quantification of the fold enrichment of N-WASp activity at invadopodium precursors in WT and 3YF cell lines. n = number of invadopodium precursors: WT (20), 3YF (25).