Mutations in BTK and PLCG2 appear under selection pressure by BTK inhibitors in REC-1 cells. (A) Circos plot summarizing the distribution of BTK and PLCG2 mutations in REC-1 cells resistant to the different covalent and noncovalent BTK inhibitors. The innermost circle represents the number of independent lines of REC-1 cells that were used for generating resistance to each inhibitor and the number of REC-1 lines that acquired the specific mutations. (B) Comparison of intracellular calcium flux between WT and mutant REC-1 cells upon stimulation with 10 μg/mL of anti-IgM. Normalization was performed for baseline [Ca²⁺]_i before anti-IgM stimulation and to maximum [Ca²⁺]_i obtained for each cell line upon treatment with ionomycin. (C) Western blotting analysis for activation of phospho-BTK, phospho-PLCγ2, phospho-AKT, and phospho-ERK upon stimulation with 10 μg/mL anti-IgM for 15 minutes. In addition, total BTK, PLCγ2, AKT, and ERK1/2 levels were analyzed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. The arrow indicates the band for GAPDH. The data shown in B and C are representatives of 3 independent measurements.