Fig. 5. Distinct CS sulfation patterns define the antibody binding specificities.

a Flow cytometry measured binding (relative GeoMFI +/− SEM) of 150 nM F4, F11, B3, C9, F8 and F3 scFv², SpyC² and B1 Fab² to A375 WT melanoma cells or with B4GALT7 knocked out (KO) (n = 3). b Inhibition of the different antibody fragments’ ability to bind ofCS with soluble CSA, CSB, or CSC (0.4–400 μg/ml) measured in ELISA competition assay. c Composition of intact ofCS and the disaccharides binding the different antibody fragments (F11, F3, F8, C9 scFv²) by footprint analysis. d C9 and F8 scFv² binding to genetically engineered CHO cells. The graphic overview (top) of the GAG biosynthesis pathways highlights the key genes studied and their assigned functions. Stars indicate genes investigated by knock-out (red) or knock-in (blue). Radar plot axis depict normalized mean fluorescence intensity of 200 nM C9 and F8 scFv² binding to cells (n = 1–3) without (purple lines) or with chABC treatment (orange lines). e Heatmap of statistically significant hits obtained by Mass Spectrometry analysis of protein pull-downs from cell supernatants with C9, F4 and F3 scFv² and SpyC² as a control run in triplicates (n = 3). Two-sided unpaired t-test with Benjamin–Hochberg correction for multiple hypotheses testing was applied to determine differentially expressed proteins (significance threshold set to 1% and fold change to 2). Missing values are colored in gray, and lo2 transformed label-free quantification (LFQ) values are row mean normalized. f Heatmap representing row mean corrected LFQ values of hits obtained by MS analysis of CSPG pull-downs from human colon biopsy (H352) with C9, F4, and F8 scFv² and SpyC² in combination with chABC treatment as indicated, prior to enrichment analyzed as in (e). g Volcano plot of human colon biopsy (H352) processed and analyzed as in (e). with selected CSPG hits highlighted in black for each protein. Source data are provided as a Source data file.