Figure 7. L1CAM is a mediator of the pro-invasive effects of FUT8.

(A) Western blot of L1CAM or FUT8 in WM3248 cells stably overexpressing L1CAM or control vector and transfected with NTC or FUT8 siRNA. L1CAM IP on WM3248 cells lysates stably overexpressing L1CAM or control vector and transfected with NTC or FUT8 siRNA. Anti-L1CAM immunoprecipitates were blotted with biotinylated LcH or α-L1CAM Ab1. (B) Trans-well matrigel invasion assay on WM3248 melanoma cells stably overexpressing L1CAM or control vector and transfected with NTC or FUT8 siRNA. n=5 fields per replicate; 5 replicates per condition. Data shown is representative of three independent experiments. (C) Trans-well matrigel invasion by MeWo melanoma cells stably overexpressing FUT8 or control vector, scale bar, 100 μm. Western blot of FUT8 is also shown. n=5 fields per replicate; 5 replicates per condition. Data shown is representative of three independent experiments. (D) L1CAM IP on whole cell lysates of MeWo cells stably overexpressing FUT8 or control vector. Anti-L1CAM immunoprecipitates blotted with biotinylated LcH or α-L1CAM Ab1. (E) Western blot of FUT8 and L1CAM in MeWo cells stably overexpressing FUT8 or control vector and transfected with NTC or L1CAM siRNA. (F) Trans-well matrigel invasion by MeWo melanoma cells stably overexpressing FUT8 or control vector and transfected with NTC, L1CAM or FUT8 siRNA. n=5 fields per replicate; 5 replicates per condition. Data shown is representative of three independent experiments. (G) Western blot of L1CAM in MeWo cell line overexpressing FUT8 or control vector, and transfected with NTC or L1CAM siRNA. Samples were blotted with α-L1CAM Ab1. (H) Western blot of cleaved and full length L1CAM on SkMel147 cells transfected with NTC or FUT8 siRNA and treated with α2-antiplasmin. Cells were transfected with siRNAs for 48 hr then incubated with α2-antiplasmin (5 μg/ml) for 16 hr. L1CAM Ab3 preferentially recognizes cleaved L1CAM fragment while L1CAM Ab1 preferentially recognizes full length L1CAM. (I) Quantitation of cleaved L1CAM (~85 kDa) using ImageStudioLite software. Tubulin was used for normalization of loading (mean ± SD of 3 replicates). (J) Western blot of cleaved and full length L1CAM on lysates from MeWo cells stably overexpressing FUT8 or control vector and treated with plasmin. Cells were cultured for 24 hr then incubated with plasmin (3 μg/ml) for 24 hr. (K) Quantitation of cleaved L1CAM. Tubulin was used for normalization of loading (mean ± SD of 3 replicates). All experiments were performed in triplicates and representative images are shown. Data are presented as mean ± SD. Two-tailed unpaired t test. *p=0.01 to 0.05, **p=0.001 to 0.01 and *** p=0.0001 to 0.001, ns: not significant. See also Figure S6.