Fig. 2. Btk SH2 domain is critical for kinase activation.

a Schematic representation of Btk constructs used in this study. Construct/domain boundaries and location of the key activating tyrosine phosphorylation sites (pY223 and pY551) are indicated. b Representative SDS-PAGE analysis of recombinant untagged Btk proteins purified from Sf9 cells. c In vitro autophosphorylation assay performed with recombinant Btk proteins at room temperature. The levels of pY551 (red channel) and total Btk (green channel) were assessed using immunoblotting in a dot blot apparatus. d Btk autophosphorylation kinetics shown in c normalized to total Btk signal and the calculated slopes of linear fits (relative velocities). Data are mean ± SD of at three independent experiments (n = 3). e Relative velocities of Btk autophosphorylation relative to KD. Data are mean ± SD of three independent experiments (n = 3) and P-values were calculated using an unpaired t-test. f HEK293 cells were transiently transfected with the indicated Btk constructs containing an N-terminal 6xMyc tag and kinase activation assessed by immunoblotting of cell lysates. g Quantification of pY551 and total pY shown in f normalized to total Btk (Myc) expression and relative to the KD. Data are mean ± SD of three biological replicates (n = 4) and P-values were calculated using an unpaired t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001. Source data are provided as a Source Data file.