Fig. 7. Rac is down-regulated on ceramide treatment in HMN1 cells. HMN1 cells were starved overnight and then stimulated with C2-ceramide (50 µM) for the indicated times. A, cells were lysed in the presence of GST-PBD and precipitated with glutathione-agarose beads. The associated, active Rac was detected by immunoblotting. Top panel, total Rac present in whole cell lysates; bottom panel, active Rac present in GST-PBD pulldowns. B, in parallel, lysates were immunoprecipitated with anti-TIAM antibody, followed by immunoblotting with the same antibody. The percentage of dead cells at each time point, as determined by trypan blue uptake, is indicated below each lane; ND, not determined. At 3 h of ceramide treatment, 43% of cells were dead.