Fig. 2. Catalytic activity of BTK variants.

COS-7 cells were transfected with wild-type BTK and the enzymatically active BTK gatekeeper variants. Thirty-six hours post transfection, the cells were serum starved for 4 h, BTK inhibitor treated for 1 h and activated for 5 min. BTK protein expression and phosphorylation of Y223 and Y551 were evaluated by immunoblotting. Numbers below bands indicate ratio of phosphorylated protein to total protein as obtained by densiometric quantification with background signal subtracted. β-actin was utilized as an internal loading control and values displayed for BTK phosphorylation were normalized to wild-type. A Gatekeeper variants inhibited at 0.5 μM of ibrutinib. B Ibrutinib-resistant variants requiring higher ibrutinib concentration. C Ratio of Y223 phosphorylated protein over total protein (as quantified by densiometric analysis) from C481S, T474A, T474S/C481S, and T474A/C481S variants. Washout (wo) was performed three times in serum-free medium prior to activation.