Visualization and characterization of GM2-rich lipid raft microdomain

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We raised a monoclonal antibody which enables the visualization of ganglioside GM2-rich microdomains as highly punctate dots on a human neuroblastoma cell, GOTO. Using this antibody, we showed that raft-like microdomains of GM2(GM2rafts) were colocalized with flotillin-2/reggie-1-positive microdomains. Prelabeling of GM2 rafts with a fluorescent probe, Alexa555. Anti-GM2, demonstrated internalization and delivery of the raft. flotillin assembly to the recycling endosomes. Depletion of cholesterol with methyl-β-cyclodextrin(MβCD) resulted in disruption and targeting of GM2 rafts to lysosomes. These results suggest that in GOTO cells, GM2 rafts are actually present in association with flotillin and undergo internalization into distinct pools of endosomal vesicles depending on their cholesterol contents.