Figure 5.

Recovery of PI4KIIα and PI 4-kinase activity on synaptobrevin/VAMP2-positive organelles by immunoisolation. Anti-synaptobrevin 2/VAMP2 Abs or control IgGs were conjugated to methacrylate beads and used for organelle immunoisolation from an LP2 fraction. (A) Electron microscopy of the material bound to control beads (Left) and to the anti-synaptobrevin 2/VAMP2 beads (Right). Arrows point to synaptic vesicles, which are not present on the control beads. (B Left) A Western blot analysis of the starting material (LP2) and of the material bound (B) and unbound (U) to the beads. Note that the nearly complete recovery of synaptophysin on the beads correlates with a similar recovery of PI4KIIα. (Right) Similar results were obtained for the PI kinase activity. The partial recovery of synaptic vesicle antigens and PI4KIIα in control IgG bead pellets is due to nonspecific sedimentation of larger membrane fragments present in LP2.