HPAECs (∼50% confluence) grown on 35-mm dishes or cover slips were transfected with either scrambled siRNA or SphK1 siRNA or SphK2 siRNA (25 nM, 72 h), then starved for 3 h in 0.1% FBS in EBM-2 without growth factors. Cells on cover slips were stimulated with 0.1% BSA-complexed S1P (1.0 µM) for 5 min, washed, fixed, permeabilized, probed with anti-Rac1 antibody (A) and anti-IQGAP1 (B) antibody, and examined by immunofluorescence microscopy using a ×60 oil objective. In C, activated Rac1 was immunoprecipitated from total cell lysates (500 µg of total protein) from control, transfected and S1P (1.0 µM) treated cells using PAK-1 PBD agarose beads as described under “Experimental Procedures”. Rac-1-GTP bound to PAK-1 PBD were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-Rac1 antibody. Shown is a representative blot from three independent experiments.