Fig 1: Loss of SEZ6 reduces HNK-1 epitope on GluK2/3 AThe glycome fingerprint of GluK2/3 was analyzed by lectin chip microarray (LecChip). Lectin PHAL and PHAE (indicated with asterisks) detected reduced amounts of the oligosaccharide Galß1-4GlcNAcß1 on GluK2/3 in SEZ6KO neurons (plot shows mean ± SEM, 3 WT replicates and 4 SEZ6KO replicates were used, discoveries were determined using the FDR method of Benjamini and Hochberg, with Q = 2%).BImmunoblot analysis revealed reduction of HNK-1 epitope on GluK2/3 in SEZ6KO brains. Left panels: Comparable amounts of GluK2/3 were immunoprecipitated (IP) in WT and SEZ6KO brains using excess of brain homogenates compared to beads. Anti-HNK-1 antibody was used for detection. HNK-1 band on GluK2/3 was detected only in WT brains but not in SEZ6KO brains (n = 6). As a control, HNK-1 modification of NCAM-1 was unaltered, as revealed after IP of NCAM-1 from WT and SEZ6KO brains (n = 3), followed by detection with anti-HNK-1 antibody. Different isoforms of NCAM-1 are annotated with arrowheads (NCAM 180, NCAM 140, and NCAM 120). Mid panel: To prove the specificity of the HNK-1 antibody, GluK2/3 was immunoprecipitated and digested with Peptide-N-Glycosidase F (PNGase F), which removes N-linked oligosaccharides. Upon PNGase F digestion, HNK-1 was not detectable in WT brains and the molecular weight of GluK2/3 was reduced (n = 2). Right panels: In total brain homogenates (input), no difference of general HNK-1 epitope or NCAM-1 levels was detected. Source data are available online for this figure.