FAK activity is required and activated by Src under stimulation. (A) FAK kinase activity FRET biosensor. The higher the activity, the lower the FRET index. (B) FRET index (averaged over the whole cell) of the stably expressed FAK biosensor in unstimulated (n = 593), 4-h HGF-treated (n = 183), PF228-treated (n = 455), PP1-treated (n = 392) cells, single cells (n = 74), and 4-h HGF-treated single cells (n = 108), confluent cells (n = 91), and 4- to 5-h HGF-treated confluent cells (n = 202). ns, not significant. (C) Top: FAK domain organization and tyrosine sites. Bottom: Typical Western blot from lysates of unstimulated and HGF-treated (3 h) with or without PP1 or PF228. (D) Phosphorylation levels FAK Y397, Y576/577, Y861 normalized to total FAK level from Western blots of cells treated as in panel c (n = 7 lysates). (E) Top: Typical micrographs of GFP-β-catenin cells with and without HGF and PF228. Bottom: Area after 2 h normalized by the area at t = 0 for unstimulated cells, HGF-treated (data from Fig. S1 I), and HGF- and PF228-treated cells (n = 10 cells). (F) GFP intensity in the nucleus (relative to cytoplasm) of GFP-β-catenin 5 h with or without HGF (50 ng/ml; data from Fig. 1 E), and with PF228 (10 µM) with or without HGF (n = +/− 37/43 cells). (G) FRET index change of EcadTSMod in cells 5 h with and without HGF (50 ng/ml; data from Fig. 1 D), and with PF228 (25 µM) with or without HGF (n = +/− 110/99 cells). (H) Top: Typical micrographs of wounded sheet migration of GFP-β-catenin cells with and without (same control as in Fig. 3 D) PF228 (25 µM). Bottom: Migration speed (mean distance traveled by cell front per hour) with or without (same control as Fig. 3 D) PF228 (25 µM; n = 121 cells). Bars: (wound healing) 100 µm; (FAK sensor) 20 µm. Two-tailed Mann–Whitney or Kruskal–Wallis tests. Values plotted are mean ± SEM.