Figure 3.

PIP₃BP and GAKIN form a complex on synthetic liposomes containing PIP₃. Binding of recombinant GST-PIP₃BP and GAKIN motor-FHA (1–557) to liposomes was examined using a membrane flotation assay. PIP₃-containing liposomes (10% PIP₃ and 90% PC) or PIP₂-containing liposomes (15% PIP₂ and 85% PC) were incubated with GAKIN motor-FHA (1–557) and GST-PIP₃BP or control GST, and liposome and soluble protein fractions were separated by sucrose gradient centrifugation. Liposomes were recovered from the top of sucrose step gradient, whereas the unbound protein remained at the bottom. GAKIN motor-FHA (1–557) and GST-PIP₃BP in the top and bottom fractions were detected by Western blotting using anti-GAKIN and anti-GST polyclonal antibodies, respectively.