Figure 4.

Cortactin phosphorylation regulates NHE1 recruitment to the invadopodia. (A) Representative images of endogenous NHE1 and cortactin colocalization. (B) Representative image of MDA-MB-231 cells transiently expressing the WT NHE1-HA and WT cortactin. (C and D) The colocalization experiments were repeated in cells expressing KRA-NHE1–WT cortactin (C) and WT NHE1–3YF cortactin (D). Insets show enlarged views of the boxed regions. (E) Quantification of the cortactin–NHE1 colocalization. Results are based on the analysis of 15 cells/group; *, P < 0.01. (F) Quantification of NHE1-positive invadopodia precursors and mature invadopodia. The fraction of cortactin–NHE1–degradation colocalization was calculated in cells expressing WT NHE1-HA and WT cortactin. *, P < 0.05; **, P < 0.01. (G) Cells expressing WT cortactin or 3YF cortactin were lysed followed by coimmunoprecipitation of cortactin and NHE1. Although the NHE1 antibodies recognized cross-reacting bands, we used specific NHE1 siRNA to determine that the major identified bands were NHE1 (Fig. S2, D and E). (H) Quantification of NHE1 and cortactin coimmunoprecipitation. Results are based on three independent experiments. *, P < 0.03. (I) Quantification of cofilin–cortactin AP FRET at mature invadopodia and invadopodium precursors in MDA-MB-231 cells treated with NHE1 siRNA and rescued with either WT NHE1 or KRA NHE1. Mock cells are NHE1KD cells nucleoporated without a DNA construct (n = 2, >15 cells per group). Error bars indicate SEM. **, P < 0.01.