Fig. 5

NHE1 co-localizes with ERK2 shown by immunofluorescence. a Immunofluorescence images of AP-1 cells (hNHE1 WT, D3-AXA and F2-AA) treated or not for 15 min with EGF (100 ng/ml). Merged images were zoomed to highlight the co-localization of ERK1/2 and NHE1 (white arrowheads). All other variants are shown in Additional file 6: Figure S6. Data are representative of three independent biological replicates. b Representative line scans across membrane areas of images as in a. Line scans were performed using Olympus image analysis software. The figure shows the pixel intensities at each wavelength over the line shown, in the absence and presence of EGF as shown. Data are examples based on analysis of at least 60 cells in three to four independent replicates per condition. c Summary of line scan analysis data for all variants. The figure shows the percentage of cells with NHE1-ERK1/2 co-localization in both membranes, based on the experiments illustrated in b. Data are shown as mean percentage with SEM error bars, based on analysis of at least 60 cells in three to five independent replicates per condition. §§ and §§§, p < 0.01 and p < 0.001, respectively, relative to AP1 + EGF; * p < 0.05 relative to own control in absence of EGF. Two-way ANOVA, Šídák’s multiple comparisons test