Fig 1: QSOX1 oxidizes Golgi glycosyltransferases Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice. Lysates were treated with PEG‐mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1.As for panel (A) but using antibodies to St3gal1.As for panel (A) but using antibodies to B3galt5.Structure of the human ST6GAL1 catalytic domain in complex with CMP (PDB ID: 4JS2; Kuhn et al, 2013) with cysteine side chains shown as spheres and numbered. Numbering according to the murine St6gal1 sequence is indicated in parentheses.QSOX1 oxidizes St6gal1 in vitro. MAb492.1 is a monoclonal antibody that inhibits human QSOX1 (Grossman et al, 2013). St6gal1 with one pair of free cysteines is modified by two PEG‐mal additions (2PEG), whereas protein with two pairs of free cysteines acquires four PEG‐mal additions (4PEG). The change in migration per two PEG‐mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs. protein (Javitt et al, 2020b).QSOX1 oxidation of St6gal1 mutants. The C350‐C361 disulfide (the redox‐active disulfide present in the C139A‐C403A mutant) is oxidized rapidly, while the C142‐C406 disulfide (present in the C350A‐C361A mutant) is oxidized slowly. Data information: For the experiments in panels (A–C), equal amounts of total protein were applied to each gel lane, and no reducing agent was added. Experiments were repeated three times with fresh new lysates, and representative blots are shown. Source data are available online for this figure.