Impaired SDF-1–induced Rac activation in MDS CD34⁺ cells. (A) MDS and normal CD34⁺ cells were stimulated with 100 ng/mL SDF-1 for the indicated time and activated Rac was precipitated using GST-PAK-CRIB and visualized by Western blotting with Rac antibodies (top). To compare the total amount of Rac protein in the samples, total lysates were analyzed by Western blotting with Rac antibodies (bottom). Three representative examples of 3 independent experiments are shown. (B) For quantification of active Rac, densitometry values of precipitated Rac were divided by the densitometry values of total Rac protein present in the same lysates. Means of 5 independent experiments are shown. Significant differences are indicated with an asterisk (P < .05). (C) MDS CD34⁺ cells (n = 4) were transduced with mock empty vector or Rac1V12. Cells were stimulated with 100 ng/mL SDF-1 for the indicated time. Cells were fixed and permeabilized, and F-actin was stained with 2.5 U/mL Alexa Fluor 594-Phalloidin. Phalloidin binding in GFP + cells (ie, transduced cells) was determined by FACS analysis and expressed as a percentage of that in unstimulated cells. (D) MDS CD34⁺ cells (n = 3) that were transduced with mock empty vector or Rac1V12 were applied to the upper compartment of a microchamber transwell system with 5-μm pores. Migration was induced by 100 ng/mL SDF-1 for 4 hours at 37 °C after which transmigrated cells were harvested from the lower compartment and counted by FACS. The assay was done in duplicate and the ratio between transmigrated cells and total amount of cells added to the upper well was calculated. The mean of 3 experiments is shown.