AKT, ERK, and NF-κB activity in AML cells is augmented by BTK inhibition. (A) AML cell lines and AML blasts were treated with 0.5 and 1 µM of ibrutinib for 8 hours and then whole-cell extracts were prepared and western blot analysis was conducted for p-p65, p65, and β-actin protein levels. (B) AML cell lines (TF-1 and U937) were transduced with BTK-targeted miRNA GFP-tagged lentiviral constructs (BTK437 and BTK1092) as well as negative control. Protein extracts were also obtained and western blot analysis was conducted for p-p65 and β-actin protein levels. (C) AML cell lines and AML blasts were treated with 0.1 to 5 µM of ibrutinib for 8 hours, and nuclear extracts were prepared and an NF-κB binding assay was performed for p50, p65, and c-Rel. (D) AML cell lines were treated with 1 µM ibrutinib for various times and then whole cell extracts were obtained. Western blot analysis was conducted for p-AKT-S473, total AKT, p-ERK and total ERK, and β-actin protein levels. (E) U937 lines were transduced with miRNA GFP-tagged lentiviral constructs (Neg-miRNA and BTK437-miRNA) for up to 8 days. Whole-cell extracts were prepared and western blot analysis was conducted for p-AKT-S473, total AKT, and β-actin protein levels. (F) Primary AML cell lines (AML #22 and AML #24) were treated with 3 to 1000 nM of ibrutinib for 8 hours. Whole-cell extracts were prepared and western blot analysis was conducted for p-AKT-S473, total AKT, p-ERK and total ERK, and β-actin protein levels.