(A) Activation of full-length Btk on membranes. Left panel: Btk (1 μM) autophosphorylates rapidly on lipid vesicles (total lipid concentration, 1 mM) containing 75% DOPC, 20% DOPS and 5% PIP3. Right panel: end-point autophosphorylation assay of Btk in the presence of lipid vesicles. The molar concentration of DOPC is kept at 75%, and the lipid fractions of PIP3 is 0%, 2%, 5%, and 10%, with the remaining lipid fraction filled with DOPS. The measurements are performed after 2 min incubation with lipid vesicles in the presence of ATP/Mg2+. (B) End-point autophosphorylation assays of Btk in solution in the presence of inositol phosphates. Upper panel: Btk (2 μM) autophosphorylates rapidly in the presence of 100 μM IP6 in solution, with the results after the first 5 min of incubation shown here. Essentially no phosphorylation is detected in Btk samples incubated with IP3, IP4 or I(1,3,4,5,6)P5, and very low phosphorylation is detected in Btk samples incubated with I(1,2,3,5,6)IP5 and I(2,3,4,5,6)IP5 in the same experiment. Lower panel: the assay is repeated with other inositol phosphates at a higher protein concentration (4 μM) and longer incubation time (10 min), and the gel was exposed for a longer time. There is a small amount of degraded Btk in the sample, as judged by the coomassie staining, leading to multiple bands in the western blot shown here and in panel C. (C) Activation of full-length Btk (2 μM) in the presence of IP6 (100 μM). (D) Activation of full-length Btk (2 μM) with increasing concentrations of IP6.