CNF_Y-mediated activation of Rho GTPases and morphologic modification and F-actin rearrangements of murine macrophages.

<p>(<b>A</b>) J774A.1 macrophages were treated with 25 nM purified CNF_Y toxin for 3 h. Toxin treated cells were lysed and aliquots of the cell lysates were taken for westernblot analysis. Remaining samples were incubated with beads-coupled with the GTPase-binding domains of effector proteins interacting with GTP-bound Cdc42, Rac1 and RhoA. The amount of the three different activated Rho GTPases and the total amount of the Rho GTPases of the lysates were analyzed with specific antibodies. (<b>B,C</b>) Fluorescence microscopy of unstimulated (−PMA) and stimulated (+PMA for 48 h) J774A.1 macrophages untreated (<b>B</b>) or treated with 10 nM purified CNF_Y toxin (<b>C</b>) for 24 h and stained with phalloidin-FITC and DAPI. The arrows indicate actin-rich membrane ruffles, filopodia and stress fibres induced by CNF_Y incubation.