L1 is present in exosomes. Rat and human glioma cells were cultured in serum-free media overnight and the media was put through 3 rounds of centrifugation as described to isolate exosomes. The resulting pellets were resuspended and analyzed by western blot with anti-L1 antibodies UJ127 and NCAM-L1. Full-length L1 was detected in exosomes released only from U87/LacZ human glioma cell line using UJ127 (A), whereas smaller weight proteolyzed forms were detected in exosomes from all 3 cell lines using NCAM-L1 (B). The 32 kDa transmembrane fragment was detected only in U-87/LacZ exosomes (B). A cell extract (435 CE) and exosome preparation (435 Ex) from human breast cancer cell line MDA-MB-435 served as a positive control for L1 detection in cells and exosomes. Exosome preps were analyzed for exosomal marker Tsg101 as well as for L1 in (C). Each exosome preparation showed the predicted 45 kDa band for TSG101.