Fig. 4. NCAM is localized in Rab4- and transferrin receptor positive endosomes. (A) Cortical neurons isolated from E15.5 mice (NCAM^+/–) or B35 cells were transiently co-transfected with NCAM140 and GFP-Rab4 or B35 cells were transiently co-transfected with either NCAM140 or NCAM180 and GFP-Rab4. Endocytosis was induced for 1 hour by application of an NCAM-specific antibody. Cells were then fixed and processed for immunofluorescence analysis by visualization of NCAM with secondary Cy3-conjugated antibodies. The experiment was carried out three times with at least 15 cells analysed in each experiment. (B) Cortical neurons from E15.5 mice (NCAM^+/–) were transiently transfected with NCAM140 and endocytosis of NCAM was induced for 1 hour by application of an NCAM-specific antibody. Cells were then fixed and processed for immunofluorescence analysis using FITC-conjugated secondary antibodies for visualization of transferrin receptors (TfR) and Cy3-conjugated secondary antibodies to detect NCAM.