BTK is highly expressed and constitutively phosphorylated in AML. (A) Control CD34⁺ cells, AML patient cells, and AML cell lines measured for constitutive levels of BTK phosphorylation by western blotting analysis, blots re-probed for wild-type BTK and β-actin to show BTK expression, and sample loading, respectively. Correlation analysis of p-BTK/BTK expression using densitometry is shown. (B) Primary CD34⁺ cells, AML cells, and AML cell lines were analyzed for phosphorylated BTK (Y223) (green) and total BTK (red) by immunocytochemistry. 4′,6-diamidino-2-phenylindole nuclear stain is shown in blue. (C) Using the immunocytochemical images captured, p-BTK was calculated as a percentage of total BTK. Values indicate the mean ± standard error of the mean from at least 5 individual experiments, sampling at least 10 representative cells from each view. *Statistical significance of P < .05 between the different treatment groups using Student t test.