Figure 6. Identification of core fucosylated glycoproteins in melanoma reveals regulators of invasion and metastasis.

(A) Schematic illustration of the experimental approach showing affinity enrichment of core fucosylated proteins by LcH lectin affinity chromatography. (B) Number of proteins identified by mass spectrometry analysis of the LcH enriched fractions of 4L, SkMel147 and MeWo membrane proteins. (C) GO enrichment analysis (category of biological processes) of common core-fucosylated proteins in three cell lines. Also see Table S2. (D) LcH affinity chromatography of whole cell lysate of 4L cells transfected with NTC or FUT8 siRNA followed by Western blot with α-L1CAM or α-NRP2 antibody. Input shows no effect of FUT8 knockdown on L1CAM or NRP2 expression. (E) L1CAM and NRP2 immunoprecipitation from whole cell lysates of 4L cells transfected with NTC or FUT8 siRNA. Anti-L1CAM or anti NRP2 immunoprecipitates were treated with or without PNGase F and blotted with biotinylated LcH or α-L1CAM Ab1 (also referred as α-L1CAM) or α-L1CAM Ab2 or α-NRP2. L1CAM Ab1 preferentially recognizes glycosylated L1CAM while Ab2 preferentially recognizes non-glycosylated L1CAM. Experiments in D and E were performed in triplicates and representative images are shown. See also Figure S5.