Figure 6.

EGF-induced recruitment of Grb2 and p85β to the membrane and Akt phosphorylation: effect of Na⁺ omission and pH. (A) Confocal images of A431 cells transfected with Grb2-SH2-YFP acquired before and after treatment for 5 min with EGF, while clamping pH_c at the indicated values with nigericin/K⁺. (B) Quantification of Grb2-SH2-YFP recruitment to the membrane in response to EGF. Data are means ± SE of 3–4 experiments. (C) Confocal images of cells transfected with p85β-YFP acquired before and after treatment for 5 min with EGF, while clamping pH_c at the indicated values using nigericin/K⁺. Arrowheads in A and C point to membrane surface not in contact with neighboring cells. Bar, 10 µm. (D) Quantification of the extent of p85β-YFP recruitment to the membrane in response to EGF. Data are means ± SE of three separate experiments. (E) Immunoblot of Akt Ser473 phosphorylation (P-Akt) in cells incubated for 5 min with EGF in Na⁺-rich buffer with or without HOE-694 or in NMG⁺-rich buffer. GAPDH was probed in the same blots to ensure comparable loading. Blot is representative of 3–4 similar experiments. (F) Quantification of the effect of NHE inhibitors, Na⁺ omission, and pH_c clamping on EGF-induced Akt phosphorylation. Data are means ± SE of ≥3 experiments of each type. *, P < 0.05; ***, P < 0.001.