FIGURE 4:

Mena interacts with PTP1B. (A) In vitro binding assay using immobilized GST-PTP1B and increasing concentrations of soluble His-Mini-Mena (containing Mena EVH1-LERER domains linked to the C-terminal coiled-coil; bottom lanes). Positive control with GST-FP4 (top lanes) and negative control with glutathione beads + GST alone (middle lanes) included to demonstrate assay specificity. Blots from representative experiment; n = 3. (B) Quantification of PLA for PTP1B and Mena in wild-type MDA-MB231 ± 1 nM EGF for 60 s. Data shown as mean ± SEM. Specificity of assay established using Mena^−/− mouse embryonic fibroblasts, where background signal was negligible (data not shown). (C) Representative images for PTP1B-Mena PLA in four breast cancer cell lines: MDA-MB231, BT549, MDA-MB453, and SkBr3. (D) Mena-PTP1B PLA across four human breast cancer cell lines compared with signal measured in wild-type MDA-MB231 cells. Data shown as mean PLA/μm² normalized to MDA-MB231 ± SEM (E) Western blot showing expression of Mena, PTP1B, and EGFR in four human breast cancer cell lines. See Supplemental Figure S4.