Analysis of CBF1-Hes1 nuclear complexes and Hes1 protein expression in B-CLL cells. (A) Nuclear extracts were prepared from freshly isolated B-CLL cells (n = 8) and PBLs and B-CD19⁺ cells from healthy donors (n = 3). CBF1-binding activity to a ³²P-labeled Hes1 probe containing 2 specific sites for CBF1 was evaluated in 10 μg nuclear proteins by EMSA. Results from all 8 B-CLL patients examined and 1 representative healthy donor are shown. (B) For competition assay, nuclear extracts from B-CLL samples were incubated with a 50-fold molar excess of unlabeled Hes1 probe or a 50-fold molar excess of unlabeled mutant Hes1 probe, 10 minutes before the addition of the ³²P-labeled specific probe. (C) For blockade of CBF1-binding activity, nuclear extracts from each B-CLL sample were incubated for 12 hours at 4°C with 5 μg anti-Notch1, anti-Notch2, or anti–β-tubulin mAbs, before the addition of the ³²P-labeled probe. In panels B and C, the data shown for patient 6 are representative of 8 patients. (A-C) Nuclear extracts (10 μg) used in EMSA were subjected to Western blot analysis with an anti-TBP mAb as a loading control. (D) Whole-cell lysates (25 μg) extracted from freshly isolated B-CLL cells (n = 25) and PBMCs, PBLs, and B-CD19⁺ cells from healthy donors (n = 5) were analyzed by Western blot for Hes1 expression. Results from 8 B-CLL patients and 1 representative healthy donor are shown. Whole-cell lysates isolated from F-9 and B-JAB cell lines were used as positive and negative controls for Hes1 expression, respectively. Protein loading was assessed by reprobing the blots with an anti–β-actin mAb. The blot of Hes1 along with the blot of Notch2, already shown in Figure 1B, were subjected to densitometric analysis and the density of the bands corresponding to Hes1 and Notch2-IC, expressed as arbitrary units, is reported for each patient. Densitometry units (U) were calculated relative to β-actin for each lane. Lane designations are identical for blots and histograms.