RhoA translocation to the plasma membrane in the reconstituted system was inhibited by prior C₂-ceramide treatment. Aliquots were taken from samples in experiments described in Figure 4. These aliquots were ultracentrifuged, and the pellets were solubilized in sample buffer to run 12.5% SDS-PAGE. Proteins were transferred, and Western blots were performed with antibody against RhoA. (A) A representative Western blot. U indicates unstimulated; G, GTPγS; D, dihydro-C₂-ceramide; C, C₂-ceramide (5 and 10 μM). (B) Scanning densitometry of the RhoA band in Western blots. Data shown are the mean ± SEM of 4 experiments. ^*Significantly different from stimulated control; P < .05.