Fig. 3. NCAM is localized only to a small extent in lysosomes. (A) Cortical neurons isolated from E15.5 mice (NCAM^+/–) or B35 cells were transiently transfected with either NCAM140 or NCAM180. Endocytosis was induced for 1 hour by application of an NCAM-specific antibody in the presence of LysoTracker (200 nM). Cells were then processed for immunofluorescence analysis by visualization of NCAM with secondary Cy2-conjugated antibodies. (B) B35 cells were transiently transfected with either NCAM140-eGFP or NCAM180-eGFP. Endocytosis was induced for 1 hour by application of an NCAM-specific antibody in the presence of LysoTracker (200 nM). Cells were then directly embedded for confocal microscopy. Experiments were carried out three times with at least 15 cells analysed in each experiment. (C) B35 cells expressing either NCAM140 or NCAM180 were mock treated or treated with chloroquine at different concentrations or with 10 µM lactacystin for 4 hours. During the last hour of incubation an NCAM-specific antibody was added to induce NCAM endocytosis. After lysis, 15 µg of total protein of each sample were loaded onto an SDS gel and subjected to western blot analysis using an NCAM-specific antibody. As loading control for the NCAM140-specific blot, all lanes were blotted with an anti-actin antibody.