Effect of HOE-694 on EGF-induced changes in pH. (A) SNARF-5F fluorescence ratio measurement of pHc. Where indicated by arrow A431 cells were stimulated with EGF in the absence (Control) or presence of HOE-694. Data are means ± SE of 3–6 experiments. (B) Top: schematic of the structure of membrane-targeted SEpHluorin/mCherry chimaera used to measure pHsm. Bottom: confocal images of SEpHluorin (left) and mCherry fluorescence (right) in A431 cells. Bar, 10 µm. (C) Representative pHsm calibration curve. Cells transfected with membrane-targeted SEpHluorin/mCherry were incubated in the presence of K+/nigericin buffers of predetermined pH. Fluorescence intensities were measured and the ratio of SEpHluorin/mCherry fluorescence is plotted as a function of pH. (D) Comparison of pHc (SNARF5-F and soluble SEpHluorin/mCherry) vs. pHsm (membrane-targeted SEpHluorin/mCherry) in cells treated with EGF for 10 min in Na+ medium in the presence and absence of HOE-694 (10 µM). Data are means ± SE of 3–5 experiments. *, P < 0.05.
