Effect of pH on activation of Rac1 and Cdc42. (A) Analysis of activated Rac1 and Cdc42 before and after treatment for 5 min with EGF, while clamping pHc at the indicated values. Active Rac1 and Cdc42 were pulled down using GST-PBD–coated beads. (B) Quantification of the effect of pHc-clamping on EGF-induced Rac1 and Cdc42 activation analyzed by GST-PBD pull-down. Data are means ± SE of three experiments. Data were compared between pHc 7.8 and pHc 6.8; *, P < 0.05. (C) Assessment of Rac1 activation by FRET imaging of genetically encoded biosensors. Activation of Rac1 was measured by FRET as detailed in Materials and methods before and after treatment with EGF, while clamping pH at pHc 6.6 or pHc 7.8. Dashed lines in whole-cell images (middle) align with the direction of protrusion and indicate the area selected for kymography and line-scan analysis (right). Bar, 10 µm. (D) Quantification of Rac1 and Cdc42 activation analyzed by FRET using line-scan analysis of the regions studied by kymography as in A. Data are means ± SE of three experiments analyzing 6–8 cells in each; ***, P < 0.001. (E) Effect of overexpression or PBD-YPet or CBD-YPet on macropinocytosis. Confocal images of cells transfected with PBD-YPet or CBD-YPet (left) incubated with EGF and TMR-Dextran (right) for 10 min in Na+-rich medium to assess macropinocytosis. Dashed lines indicate outlines of cells. Bar, 10 µm. (F) Quantification of macropinocytosis in untransfected or in highly transfected cells by measuring TMR-Dextran fluorescence intensity (right). Data are means ± SE of three experiments. Data were compared between untransfected and transfected cells; ***, P < 0.001.
