Fig. 2. Distribution of NCS-1 and PI4Kß in subcellular fractions of rat brain. (A) Differential centrifugation of rat brain total homogenates. Equal amounts of proteins (12 µg) from each fraction (defined as described in Materials and Methods) were analyzed by SDS-PAGE and western blotting. The blots were probed with antibodies directed against PI4Kß (PI4K, diluted 1:4000), NCS-1, synaptophysin (Syn, diluted 1:2000) or synaptotagmin (Syt, diluted 1:1000). Note the presence of small amounts of NCS-1 and PI4Kß in a fraction of purified synaptic vesicles (SG-V). Both proteins are present in large amounts in P3 containing membrane-bound organelles of perikarya. (B) Velocity sucrose gradient centrifugation of the P3 (see A). Equal volumes (250 µl) of each fraction of the velocity gradients (fraction 1=top) were analyzed by western blotting using antibodies raised against PI4Kß (PI4K), NCS-1, calreticulin (Cal, diluted 1:2000), ribophorin (Ribo, diluted 1:250), TGN38 (diluted 1:1000) and syntaxin 1 (Syx, diluted 1:4000). (C) Fractionation of the S2 on discontinous sucrose density gradients. Equal amount of proteins from fractions 1, 2 and pellet (3) were analyzed by immunoblotting using anti-NCS1, PI4Kß, ribophorin, TGN38 and GOS-28 (GS28, 1:100) antibodies.