Fig. 1: Expression and catalytic activity of BTK gatekeeper variants in COS-7 cells.

Thirty-six hours post transfection, the cells were serum starved for 4 h and subsequently activated with serum and pervanadate for 5 min at room temperature. The cell lysates were immunoblotted for total and tyrosine (Y) 223 phosphorylated BTK protein. For densiometric quantification, background signal was subtracted and β-actin utilized as an internal loading control as well as for normalization of total BTK. Values displayed for BTK phosphorylation were normalized both to total protein and wild-type BTK. Four categories were used to quantify the relative BTK activation of the gatekeeper variants compared to wild-type, low (<0.5-fold), intermediate low (0.51–1.0-fold), intermediate high (1.01–1.5-fold) and high (>1.51-fold compared to wild-type). Two-way ANOVA, 95% confidence interval, p value ⁽*⁾ < 0.05 and **** < 0.0001.