Talin recruits NHE-1 to invadopodia and promotes NHE-1–dependent alkalinization of the intracellular space at invadopodia. (A) Coimmunoprecipitation of NHE-1 with talin. Cells were starved for 16 h and stimulated with 2.5 nM EGF. Endogenous talin was immunoprecipitated from MDA-MB-231 cell lysates using the 8d4 talin antibody, and the resulting immunoprecipitates were subjected to Western blotting with anti–NHE-1 and talin antibodies. IgG served as a negative control; three independent experiments. (B and C) Talin regulates NHE-1 localization to invadopodia. (B) MDA-MB-231 cells were stained with phalloidin (F-actin), NHE-1, and cortactin. Inset shows magnified image of invadopodia in the box. Red arrowheads denote invadopodia. Bars: (main panel) 10 µm; (inset) 1 µm. (C) Quantification of NHE-1 enrichment at invadopodia in control and talin siRNA-treated cells transfected with GFP or a series of GFP-tagged talin constructs. n > 47 invadopodia; three independent experiments. *, P = 0.034; **, P < 0.029. (D) Quantification of mean intracellular pH at invadopodia (BCECF pH biosensor). n > 56 invadopodia; three independent experiments. **, P = 0.012. Error bars represent the SEM.