Extended Data Fig. 3. Fucosylated mass spectrometric analysis and knockdown efficiency of H2K1 and H2EB1.
(a) (left) Schematic for proteomic analysis of fucosylated proteins in human WM793 melanoma cells using pLenti-GFP empty vector (EV)-, pLenti-FUK-GFP (o/e)-, or shFUK-expressing WM793 cells from8. Click chemistry biotinylated-fucosylated proteins that were pulled down using Neutravidin beads from the 6-alkynyl-L-fucose-labeled cells were subjected to LC-MS/MS, and hits were subjected to the indicated filtering scheme followed by Ingenuity Pathway Analysis (Qiagen). (right) Top 20 pathways, plasma membrane- and immune-related proteins identified by Ingenuity Pathway Analysis (Qiagen) to be significantly altered by fucosylation. (b) qRT-PCR analysis confirming knockdown of H2K1 (shH2K1; left) or H2EB1 (shEB1; right) using 2 shRNAs per target compared to control non-targeting (shNT) shRNA. n = 3 biologically independent experiments. Red arrows indicate the specific shRNA clones used in functional experiments in the remainder of the study. All error bars represent standard error of the mean.