Hypermorphic mutation R665W acquires resistance to ibrutinib and functions independently to BTK. (A) Downstream phospho-protein activation upon 0.5 μg/ml α-IgM stimulation for 15 minutes was examined by western blots in comparison with WT PLCγ2 to R665W mutation in DT40 cells. (B) 293T cells were retrovirally transduced with WT PLCγ2 or R665W mutation. The production of IP₃ upon 150 ng/ml epidermal growth factor stimulation was measured by IP₁ (surrogate of IP₃) accumulation using IP-One ELISA Kit (n = 3 repeated experiments). (C) Calcium flux in BTK-deficient DT40 lines introduced with WT PLCγ2 or R665W mutant were examined. Data represents the AUC from 6 replicates upon 3 μg/ml α-IgM stimulation, and (D) downstream phospho-protein activation was examined by western blots. DMSO, dimethylsulfoxide; KO, knockout; Un., untreated.