Effect of soluble Jagged1 ligand and GSI treatment on NF-κB activity, c-IAP protein expression, and caspase activation. Freshly isolated B-CLL cells (n = 3) were cultured for different times in 2 different conditions: for 24 hours in culture tubes at a density of 2 × 106/mL (3 mL/tube) with GSI 5 μM or DMSO as control (A,C,E), or for 48 hours in 96-well plates at a density of 106/mL (100 μL/well) on immobilized soluble Jagged1 ligand or IgG-Fc as control (B,D,F). NF-κB–binding activity to a 32P-labeled specific probe was evaluated by EMSA in 5 μg nuclear proteins extracted from the indicated cell populations (A,B). Nuclear extracts (5 μg) used in EMSA were subjected to Western blot analysis with an anti-TBP mAb, as a loading control (A,B). c-IAP2 and XIAP expression (C,D), caspase-3 processing, and PARP degradation (E,F) were analyzed by Western blot in 30 μg whole-cell lysates. Protein loading was assessed by reprobing the blots with an anti–β-actin mAb. The data shown for patient 6 are representative of 3 patients.
