Alexa 594 labeled g actin

The incorporation of Alexa 594-labeled G-actin (Invitrogen) into saponin-permeabilized cells has been described previously [12 (link), 46 (link)]. Briefly, confluent monolayers of Caco-2 were equilibrated at 25°C for 10 min, then lightly permeablized with 0.2 mg/mL Saponin in permeabilization buffer (138 mM KCl, 4 mM MgCl₂; 20 mM HEPES [pH7.4]). Alexa 594-labeled G-actin (0.45 µM; Invitrogen) was added to the cells in permeabilization buffer to favor barbed-end incorporation and was incubated at 25°C for 7 min. The cells were then fixed on ice in 4% PFA in 1×cytoskeletal stabilization buffer containing 0.2% Triton X-100 and Alexa 488 phalloidin for 1 hr.