Fig. 9. NCAM is internalized by clathrin-dependent and caveolae-dependent pathways. (A) NCAM140 or NCAM180-expressing B35 cells were preincubated with MDC (300 µM, 10 minutes), nystatin (NYS, 50 µg/ml, 1 hour) or both inhibitors together (MDC 10 min, NYS 1 hour). Endocytosis was induced for either 30 (NCAM140) or 60 minutes (NCAM180). Cell surface NCAM was detected using Cy3-conjugated secondary antibodies and internalized NCAM using Cy2-conjugated secondary antibodies. Data are the mean ±s.e.m. of three independent experiments with at least 15 cells analysed in each experiment. *P<0.05, **P<0.005, ***P<0.001. (B) Representative images of endocytosed NCAM140 or NCAM180 in B35 cells in the presence or absence of MDC and nystatin. (C) Cortical neurons isolated from E15.5 mice (NCAM^+/–) were transiently transfected with NCAM140 or NCAM180 cDNA. Cells were treated with MDC and nystatin as described in A and endocytosis was induced for 30 minutes. Cell surface and internalized NCAM was detected as described in A. Data are the mean ±s.e.m. of three independent experiments with at least 15 cells analysed in each experiment. *P<0.05, **P<0.005, ***P<0.001. (D) Representative images of endocytosed NCAM140 or NCAM180 in cortical neurons in the presence or absence of MDC and nystatin.