Figure 2.

Rho/Rock1-dependent ezrin dephosphorylation/downregulation regulates UA-mediated DISC formation and apoptosis in U937 cells. (a) U937 cell were treated without or with 20 μℳ UA for 6, 12 and 24 h. Total cellular extract were prepared and subjected to western blot analysis using antibodies against phospho-Ezrin^tyr353(p-Ezrin), Ezrin, Moesin. A densitometric analysis of the p-Ezrin and Ezrin levels were performed using the Quantity One software (Bio-Rad) and p-Ezrin/Ezrin ratio was evaluated. The values obtained represent the means±s.d. for three separate experiments. (b) Active Rho-GTP were pulled down by association with the Rho-binding domain of rhotekin at the indicated times after UA treatment, the bead/protein complexes and total Rho in lysates were detected by immunoblotting using antibody against Rho. (c) Total protein lysates were prepared and subjected to western blot analysis using antibody against ROCK1. (d) Cell lysates of control and UA-treated cells were prepared and subjected to immunoprecipitation using antibody against Fas. The associated Ezrin, Caspase-8, FADD and Fas were determined using immunoblotting. (e) U937 cells were either untreated (Control) or treated with 20 μℳ UA for 3, 6 and 12 h. Cells were collected and stained with anti-Fas antibody, followed by Alexa 488-conjugated goat anti-mouse antibody (green fluorescence for Fas); and with anti-ezrin antibody followed by Alexa 647-conjugated donkey anti-rabbit antibody (red fluorescence for ezrin). Immunofluorescence was visualized using a laser confocal scanning microscope. The merge in yellow represents colocalization between Fas and Ezrin. Scale bar represents 10 μm. Two additional studies yielded equivalent results. CF, cleavage fragment.