(A) In vitro phosphorylation inhibition assay for Abl enzyme in the presence of WBZ_4 (pink) or imatinib (blue). Active recombinant Abl enzyme (1 μg/ml) and its substrate (Abl-tide; 1 μg/ml) were incubated for 1 hour at 37°C in the presence of various WBZ_4 or imatinib concentrations. ATP (100 nM) was added to the reaction mixture. Phosphorylation of Abl-tide peptide was detected by incubation in consecutive order with anti-rabbit phospho–Abl-tide antibody and anti-rabbit HRP antibody. TMB was added to initiate the chromophore reaction, and 2 minutes were allowed for color development. The reaction was terminated by the addition of 1 M H₂SO₄. Phosphorylation of the substrate was quantified as absorbance units (AU) by spectrophotometry at 450 nm. Values obtained with the enzyme without the inhibitors (WBZ_4 or imatinib) were assumed to represent 100% phosphorylation and were compared with the values obtained with the addition of the inhibitors. (B) In vitro phosphorylation inhibition assay for C-Kit in the presence of WBZ_4 (pink) or imatinib (blue). Active recombinant C-Kit kinase (25 ng/ml) and its substrate Poly(Glu4-Tyr) (150 nM) were incubated for 1 hour at 37°C in the presence of various WBZ_4 or imatinib concentrations. ATP (100 nM) was added to the reaction mixture. Phosphorylation of Poly(Glu4-Tyr) peptide was detected by incubation in consecutive order with anti-phosphotyrosine antibody and anti-rabbit HRP antibody.