BTK phosphorylation is minimally affected by ibrutinib treatment in B cells from C481S mice. (A) Western blot on whole-cell lysates from B cells with indicated genotype (wild-type or C481S), inhibitor treatment (ibrutinib or RN486) and stimulation (anti-IgM and H₂O₂). Numbers above bands indicate densiometric quantification. *Value could not be calculated. (B) Ratio of phosphorylated protein over total protein (as quantified by densiometric analysis) from splenocytes with indicated genotype, in vivo ibrutinib treatment, and in vitro stimulation (anti-IgM and H₂O₂). For in vivo ibrutinib treatment, mice were given ibrutinib (0.3 mg/mL) in the drinking water for 1 week. Each dot represents data from an individual animal. Error bars indicate mean and SD; the unpaired 2-tailed Mann-Whitney U test was used to calculate significance (*P < .05); (dashed line) indicates significance calculated after exclusion of the outlier. For densiometric quantification in panels A-B, background signal was subtracted, β-actin used as an internal loading control and values displayed normalized to the mean protein ratio in wild-type cells.