Fig. 1.

L1 was expressed and proteolyzed in a human GBM cell line and surgical cells. a RT-PCR to detect L1 mRNA. T98G cell line exhibited the expected 461 bp band and U-118 MG showed the 461 bp band to a much less extent. The human L1 cDNA (hL1cDNA) was used as positive control. No-reverse transcriptase was used as a negative control. b Western blot analysis of L1 expression and proteolysis. NCAML1 antibody recognized the ~220 kDa full length and the ~32 kDa L1 bands in T98G cells. L1 protein expression was undetectable in U-118 MG cells. UJ127 antibody detected the full length L1 in surgical GBM21 cells. QT6 cells transfected with human full length L1 (QT6/hFL1) was used as positive control (PC) and mock transfected cells (QT6/mock) served as negative control (NC). c Western blot analysis demonstrated ADAM10 expression in T98G and U-118 MG cells, with unprocessed premature ADAM10 (~80 kDa) and the processed mature ADAM10 (~60 kDa band) in both cell lines. Jurkat cell extract was used as positive control. d Western blot analysis of cell culture medium versus cell lysate. T98G cell culture medium (under “Culture Medium” bar) showed a UJ127-positive band (~180 kDa) smaller than 220 kDa full-length band expressed by QT6/hFL1 cell lysate (PC). Mock transfected QT6 cell medium (NC) and medium from U-118 MG cells were negative controls. NCAML1 did not detect the full length L1 in the T98G cell culture medium as expected. e T98G cells immunostained for L1 and viewed by widefield fluorescence microscopy. Live cell staining using UJ127 antibody (upper panel) showed cell surface L1. Fixed cell staining using NCAML1 antibody (lower panel) detected intracellular as well as cell surface L1. QT6/hFL1 and QT6/mock cells were used as positive and negative controls. Nuclei were stained with bisbenzimide (lower row in each panel). Bar = 50 μm