RhoA translocation to the plasma membrane in intact and streptolysin O–permeabilized PMNs treated with C₂-ceramide (C₂). Intact PMNs were activated with 100 nM FMLP (F), whereas permeabilized PMNs were treated with 10 μM GTPγS (G). Cells were disrupted by probe sonication, and cytosol was separated from membranes by ultracentrifugation. Membrane pellets were solubilized in sample buffer to run 12.5% SDS-PAGE. Proteins were transferred, and Western blots were performed with antibody against RhoA. U indicates unstimulated. Results are representative of 4 experiments.