Figure 1.

Deletion of the two membrane-proximal FNIII domains in L1cam is required to generate a proteinase resistant variant. (a) Diagram of WT L1cam and mutants with the two membrane proximal fibronectin type III (FN) domains deleted alone or in combination as indicated (b) HEK293T cells were transfected with plasmids encoding WT L1cam or deletion variants alone or in combination with ADAM10 as indicated. The extend of shedding of soluble L1cam into cell culture media was assessed by Western blotting using an antibody targeting the N-terminal of L1cam, membrane staining for total protein was used as loading control (2 top lanes). Expression of L1cam and ADAM10 in cell lysates was assessed by Western blotting, using antibodies targeting the C-terminal c-myc tag (L1cam), or ADAM10. Actin was used as loading control (3 bottom lanes). (c) The amount of soluble fragments in the culture medium was quantified and displayed relative to the amount of soluble fragments from cells co-transfected with WT-L1cam and ADAM10. Mean values +/− SEM for three independent experiments are plotted (Supplementary Fig. 11). Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test. (d) HEK293T cells were transfected with plasmids encoding WT L1cam, deletion variants or an empty vector as indicated. The expression of the different variants on the cell surface was assessed by flow cytometry using an antibody targeting the N-terminal of L1cam. Example histograms are displayed. Mean fluorescence intensities for three independent experiments are displayed in Supplementary Fig. 1.