Activity-dependent localization of Rac1. (A,B) WT LDBM cells were transduced with EGFP–Rac1, EGFP–Rac1V12, or EGFP–Rac1N17 retroviral vectors. Sorted EGFP⁺/c-Kit⁺ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and stained with rhodamine-labeled phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). The percentage of cells with membrane bound Rac1 is shown as the mean plus or minus SD; n = 3. (C) Rac1 localizes to lipid rafts in response to SDF-1α treatment. WT LDBM cells were transduced with EGFP-Rac1 retroviral vector. Sorted EGFP⁺/c-Kit⁺ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and then stained with Alexa Fluor 550–labeled CTxB (red). (D) Negative effect of MβCD on Rac1 localization and F-actin polymerization. WT LDBM cells were transduced with EGFP-Rac1. Sorted EGFP⁺/c-Kit⁺ cells were treated with 5 mM MβCD for 30 minutes to deplete cholesterol followed by the addition of SDF-1α (100 ng/mL) for 30 seconds. The cells were fixed and stained with rhodamine-labeled phalloidin or Alexa Fluor 550–labeled CTxB. The representative images from 3 independent experiments are shown.