Cofilin phosphorylation, PI(4,5)P₂ hydrolysis, and actin FBE formation. (A) Analysis of cofilin phosphorylation from lysates of A431 cells before and after treatment for 1 or 3 min with EGF in Na⁺-rich buffer. Blot is representative of three experiments. (B) Assessment of PI(4,5)P₂ hydrolysis during EGF stimulation. PI(4,5)P₂ was monitored using PLC∂-PH-GFP and confocal imaging. Recording was initiated upon addition of EGF and TMR-dextran in Na⁺-rich buffer. During the initial 6 min, only green fluorescence was monitored. After 6 min excess TMR-dextran was washed and formed macropinosomes were visualized in red channel. Insets show magnifications of indicated areas. (C) Quantification of PLC∂-PH-GFP localization to the plasma membrane during EGF stimulation. Data are means ± SE of three separate experiments. (D) Detection of actin FBEs by imaging rhodamine-actin in cells before and after treatment for 1 min with EGF in Na⁺-rich buffer. Images are representative of three experiments. Insets show regions at the edge of cells typically selected for quantification. (E) Quantification of FBE cells before and after treatment with EGF in Na⁺-rich buffer or in pH_c clamping medium. Data are means ± SE of three separate experiments. The significance of the difference ± C. difficile toxin B (Tox B) was calculated using Student’s t test; ***, P < 0.001.