Over-expressing L1-ectodomain in MDA-MB-468 cells. (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).