Figure 3.

Subcellular distribution of PI4KIIα and lipid kinase activities. (A) Western blot analysis of subcellular fractions from a rat brain homogenate (H) with Abs directed against PI4KIIα and other neuronal proteins. Fractions were prepared according to Huttner et al. (30). The P2 fraction represents crude synaptosomes, which after lysis yield quickly and slowly sedimenting membrane fractions denoted LP1 and LP2, respectively. The LP2 fraction, which is highly enriched in synaptic vesicles (crude synaptic vesicle fraction), also contains a large pool of PI4KIIα. Note the absence of synaptophysin (a synaptic vesicle membrane marker) and PI4KIIα in the LS2 fraction, which contains only soluble proteins. (B) TLC analysis of ³²P incorporation into PIP and PIP₂ after incubation of the fractions with a brain phosphoinositide mixture, [³²P]ATP, and Triton X-100. X denotes an unidentified spot.