Cortactin interacts with cofilin at invadopodium precursors through a phosphorylation-dependent mechanism. (A) Representative cofilin-cortactin FRET efficiency image of a resting cell (0 s EGF). Red = cortactin, green = cofilin. Box indicates bleached area. Bottom right inset shows close up of FRET efficiency between cofilin and cortactin at invadopodium precursors. Bar, 10 μm. (B) Quantification of FRET between cofilin and cortactin at invadopodium precursors in response to EGF normalized to 0 s (FRET efficiency at 0 s = 11.8% ± 1.1). P values are compared with 0 s unless indicated. n = number of invadopodium precursors: 0 (25), 60 (24), 180 (22), 300 (17); three independent experiments. (C) Quantification of cofilin-cortactin FRET at invadopodium precursors in starved MTLn3 cells lines expressing WT, 3YF, and 3YE cortactin with endogenous cortactin knocked down normalized to WT (FRET efficiency of WT cortactin = 4.2% ± 0.36). P values are compared with WT. n = number of invadopodium precursors: WT (22), 3YF (22) 3YE (17); three independent experiments. (D–G) IP experiments between cofilin and cortactin in response to EGF. (D) Representative Western blot showing IP of cofilin and coIP of cortactin. (E) Quantification of the protein level of Co-IP cortactin/IP cofilin normalized to 0 s. n = number of independent experiments: 6. P values are compared with 0 s (F) Representative Western blot showing IP of cortactin and coIP of cofilin. (G) Quantification of the protein level of the Co-IP cofilin/IP cortactin normalized to 0 s. P values are compared with 0 s. n = number of independent experiments: 4. (H) Representative Western blot showing IP of cofilin and coIP of cortactin in cortactin WT vs. 3YF cell lines treated with cortactin siRNA. (I) Quantification of the protein level of Co-IP cortactin/IP cofilin normalized to WT. n = number of independent experiments: 3.