Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

Analysis ofGranin Aggregates in the TGN ofPC12 Cells (Standard Procedure) Preparation of ?GN Vesicles. PC12 cells pulse labeled for 5 min with [35 S]sulfate were placed on ice to stop intracellular transport . All subsequent steps were performed at 0-4°C. Cells (two 15-cm dishes of subconfluent cells) were homogenized and a postnuclear supernatant (N1 .2 ml) was subjected to velocity sucrose gradient centrifugation as previously described (lboze and Huttner, 1990) . After velocity centrifugation, fractions (1 ml) were collected from the top of the gradient . In some experiments, 100-141 aliquots of these fractions were analyzed by SDS-PAGE followed by fluorography. Fractions 8-10, which contained the peak of ["S]sulfatelabeled granins and which have been shown to contain [35 S]sulfate-labeled TGN vesicles (Tooze and Hutmer, 1990) , were pooled, slowly diluted with an equal volume of 10 mM Hepes-KOH, pH 7.2, and divided into aliquots (three per gradient) .