In vivo lenalidomide treatment of primary human CLL cells results in decreased RhoH expression and increased RhoA and Rac activation. CLL patients were treated daily for 8 days with 2.5 mg lenalidomide within a clinical phase 1 study. All samples obtained from 6 patients before (pre) and on day 8 (d8) of lenalidomide treatment contained > 87% CD19⁺ cells. Representative blots of 3 patients are displayed. (A) Western blot analysis for RhoH protein expression. (B-C) RhoA and Rac activities determined by pull-down assays. In each panel the protein of interest or active (GTP-bound) protein is shown in top panel and the loading control or total protein is shown in the bottom panel. The bar graphs in the middle panel show ratio of RhoH to β-actin and activated GTP-bound RhoA or Rac protein to total RhoA or Rac protein based on densitometric measurements of the respective Western blot band before and after treatment for each of the 3 patients. The bar graphs in the bottom panel summarize the ratios obtained in all 6 patients (gray bars: pre treatment vs black bars: day 8 results; mean ± SEM, *P < .05, Student t test, n = 6).