Fig. 3. Intracellular distribution of CgA, CgB, lamp1 and lamp3 in CV-1 fibroblasts (A,B,C); expression of CgA in PC12-64 and CV-1 cells (D). A-C show merged images of CV-1 cells stably transfected with human CgA and dually immunodecorated for the latter granin (monoclonal LK2H10, red) together with (green) lamp1, lamp3 or CgB (monoclonal CIRO antibody), respectively. Notice that CgA and the two lysosomal markers, lamp1 and lamp3, which are largely dissociated from each other in the GC/TGN perinuclear area, do co-localize (yellow) in most of the discrete puncta scattered in the rest of the cytoplasm (A,B). Also note that expression of CgA does not induce any co-appearance of CgB (C). Scale bars: 8 µm. D shows western blots (25 µg/lane) of different cell lines expressing (PC12-15; PC12-64) and not expressing (CV-1) endogenous CgA, some of which were also analysed after stable transfection with human CgA (hCgA PC12-64; hCgA CV-1). The same blot was immunostained with the polyclonal anti-CgA antiserum GE-19 (a); with the monoclonal anti-hCgA antibody LK2H10 (b); and with the polyclonal anti-CgB antiserum PE-11 (c; similar results were obtained with the monoclonal CIRO). Notice that GE-19 recognizes both rCgA (86-100 kDa) and, to a minor extent, also hCgA (70-63 kDa), whereas LK2H10 recognizes hCgA only. In PC12-64 cells expression of the transfected CgA is accompanied by a decrease of the endogenous granin. M_r of protein standards is indicated to the right in kDa.