Figure 6.

Identification of fucosylated glycoproteins in ESCC revealed regulators of invasion and metastasis. (A) Schematic illustration of the experimental approach showing affinity enrichment of core-fucosylated and α-1,2-fucosylated proteins by LCA and UEA-I lectin affinity chromatography respectively. (B) The effect of enriched fucosylated protein by UEA-I lectin with or without α-1,2-fucosidase on KYSE150 and KYSE450 cell invasion. (C) Coomassie brilliant blue (CBB) staining of gels of whole cell lysate proteins and LCA enriched fucosylated proteins in TSTA3-WT and control group. (D) Number of overlapping proteins between lectin enriched proteins identified by in gel mass spectrometry analysis and differentially expressed glycoproteins in N-glycoproteomics data. (E) LCA affinity chromatography of whole-cell lysates of ESCC cells transfected with TSTA3-WT and NC followed by western blot with LAMP2 antibody. (F) A representative western blot of LCA-affinity purified LAMP2. LCA-affinity purification was done in the absence or presence of various concentrations of α-L-fucose (0, 6, 30 and 60 mM). (G) LAMP2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-LAMP2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated LCA or LAMP2 antibody. (H) UEA-I affinity chromatography of whole-cell lysate of ESCC cells transfected with TSTA3-WT and NC followed by western blot with ERBB2 antibody. (I) A representative western blot of UEA-I-affinity purified ERBB2. UEA-I-affinity purification was done in the absence or presence of various concentrations of α-L-fucose. (J) ERBB2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-ERBB2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated UEA-I or ERBB2 antibody.