Western blot analysis of the colocalization of CXCR4 with Rac-1 in various fractions of cell membranes. Membranes enriched in lipid rafts (fractions 3-5) and depleted of lipid rafts (fractions 9-11). (A) Hematopoietic THP-1 cells were stimulated with fibronectin (2 μg/mL) or fibrinogen (4 μg/mL) or were not stimulated (control). CXCR4 and Rac-1 were detected in these membrane fractions by Western blot along with ganglioside GM1, a marker of lipid rafts. Experiments were performed 3 times with similar results. (B) In control experiments, cells were pretreated for 1 hour with 10 mM MβCD. A representative study is shown. (C). Detection of GTP-bound (active) form of Rac in THP-1 cells nonstimulated (–) or stimulated with SDF-1, fibrinogen (FG), or SDF-1 + FG (left panel) or with SDF-1, fibronection (FN), or SDF-1 + FN (right panel). Activated Rac was detected in lysates of stimulated cells by using Western blot, as described in “Patients, materials, and methods.” Experiments were performed 3 times with similar results. Representative results are shown.