Fig. 4. Expression, distribution and release pattern of CgA in PC12-15 and in transfected CV-1 cells. A,B show groups of PC12-15 cells immunolabelled for CgA before (A) and after (B) three hours of treatment with cycloheximide (10 µg/ml): the protein synthesis blocker had no appreciable effect on the levels and distribution of the granin. C,D illustrate the same experiment in CV-1 cells transfected with CgA. Before the treatment the cells showed accumulation of the granin in the GC/TGC area and in multiple discrete puncta scattered in the cytoplasm (C). After exposure to cycloheximide the signal was reduced to a faint trace (D). The images shown are 3D reconstructions of focal plane series comprising the whole cell thickness. Exposure conditions were the same for controls and cycloheximide-treated cells. Scale bars: 7 µm. (E) results of pulse-chase experiments showing the release pattern of huCgA from transfected CV-1 cells during a chase time of 3 hours. The percentage of released CgA measured following immunoprecipitation of the protein from the cell media and lysates at the different time-points was calculated over the total CgA (released + intracellularly retained).