The agammaglobulinemia-linked kinase, BTK, inhibits Wnt–β-catenin signaling. ( A ) NALM-6 B cells stably expressing BAR upstream of firefly luciferase and expressing a constitutive reporter that drives Renilla luciferase were transiently transfected with the indicated siRNA oligonucleotides. Forty-eight hours after transfection, the cells were treated with either WNT3A–conditioned medium or control medium. The following day, amounts of firefly luciferase were quantified, normalized to that of Renilla luciferase, and all data were plotted as fold change over untreated control siRNA. Error bars represent standard deviation from the mean for three replicates. These data are representative of three independent experiments. ( B ) Top: NALM-6 B cells were transfected with siRNAs targeting the indicated mRNAs. Two days after transfection, the cells were incubated overnight with WNT3A–conditioned media. Cell lystates were Western blotted with antibodies against BTK, CTNNB1, and TUBB1. Bottom: BTK does not decrease the abundance of CTNNB1 in unstimulated or WNT3A–stimulated cells. Patient-derived B cells were treated overnight with recombinant WNT3A at the indicated concentrations, lysed for protein analysis, and subjected to Western blotting for CTNNB1 and TUBB1. ( C ) Control B cells from normal patients were treated with recombinant WNT3A (100 ng/ml) overnight and processed for qPCR to show that Wnt–β-catenin signaling is intact in normal B cells. ( D ) Untreated B cell lines derived from patients lacking functional BTK and wild-type control B cells were processed for qPCR, showing that Wnt–β-catenin signaling is elevated in the absence of stimulation in BTK-deficient B cells. In both (C) and (D), the B cell cDNA was amplified with primers targeting GAPDH ( glyceraldehyde-3-phosphate dehydrogenase ) and the Wnt–β-catenin targets AXIN2 and CCND1 . The number of copies of AXIN2 and CCND1 were averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. These data represent three independent replicates in three independent experiments.