Fig 1: Glycoproteomic analyses reveal novel a2,6-sialylation targets of ST6GAL1. A, Experimental design of glycoproteome profiles of SNA-bound a2,6-SA+ membrane proteins isolated from ST6WT and KO cells. The membrane fractions were isolated, and a2,6-SA–linked proteins were coimmuno­precipitated with SNA-conjugated agarose beads. Purified proteins were loaded to the glycoproteomics analysis. B, The list of top ST6GAL1 target glycoproteins including their a2,6-sialylation sites on the peptide. C, Immunoblots of the ST6GAL1 substrates PODXL, ECE1, CD97, ALCAM1, and ICAM1 with immunoprecipitation (IP) by SNA (a-2,6-SA+ proteins) or agarose controls, and input controls of the membrane fraction (Mem) or whole-cell lysate (WCL) from ST6WT (+) and ST6KO (-) MDA-MB-231 cells. D, Immunoblots of ST6GAL1 target proteins and the loading control ß-actin from the lysates of ST6KO MDA-MB-231 cells transfected with the scramble control (scr) and siRNAs for PODXL, ECE1, CD97, ALCAM1, and ICAM1. E and F, Representative images of clusters at 2 hours (E) and cluster formation curves over the time (0–16 hours; F) of ST6KO MDA-MB-231 cells after downregulation of indicated genes. P scrST6KO vs. WT < 0.0001, P scrST6KO vs. siPODXL = 0.0001, P scrST6KO vs. siECE1 = 0.0175, P scrST6KO vs. siALCAM1 = 0.0260, P scrST6KO vs. siCD97 = 0.0043, and P scrST6KO vs. siICAM1 = 0.0003; scale bars = 100 µm. P values were calculated with the t test using GraphPad. Data are represented as mean ± SD of 12 experimental replicates. Cluster videos are available as Supplementary Videos S8–S14. G, Representative images of PODXL staining on patient CTCs (CD45-CK+DAPI+) via CellSearch (left) and flow cytometry–based quantification (right) of PODXL expression. H, Schematic (left) and quantification (right) of cell-binding analyses (ST6WT/ST6KO) with PODXL and a2,6-desialylated PODXL isolated from ST6WT and ST6KO cells, respectively. P values were calculated with the Student t test using GraphPad, and data are represented as mean ± SD of 3–5 experimental replicates unless otherwise indicated.