In vitro lenalidomide treatment of primary human CLL cells results in decreased RhoH expression and increased RhoA and Rac activation. Primary human CD19⁺CLL samples were treated with vehicle or 0.5μM lenalidomide for the indicated time points. CLL content was ≥ 90% in all cases. One representative experiment of 5 is shown. (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 bands. The bar graphs in the bottom panel summarize the respective ratios obtained in all 5 patients (gray bars: vehicle versus black bars: lenalidomide treatment; mean ± SEM; (A) P = .09; (B) *P = .002; (C) P = .24, Student t test, n = 5).