Effect of Notch2 down-regulation on B-CLL cell survival. Freshly isolated B-CLL cells were nontransfected (NT), mock-transfected (no siRNA), or transfected with 0.5 μM control nontargeting or Notch2 siRNA (n = 6) as described in “siRNA nucleofection” and then cultured in complete medium for 72 hours. (A) Cell viability was evaluated by annexin V/PI staining, and the results from each patient are presented as the percentage of annexin V⁻/PI⁻ cells. (B) Notch2 protein expression was analyzed by Western blot in 25 μg whole-cell lysates from the indicated cell populations, and protein loading was assessed by reprobing the blots with an anti–β-actin mAb. The results shown for patients 4, 6, and 8 are representative of 6 patients. (C) For each sample, blot lanes corresponding to control nontargeting and Notch2 siRNA were subjected to densitometric analysis and normalized to β-actin levels. Columns represent the percentage inhibition of Notch2-precursor, Notch2-TM, and Notch2-IC induced by Notch2 siRNA compared with control siRNA.