Journal: The Journal of Biological Chemistry
Article Title: PA14 domain of glycosyltransferase B4GALNT3 is a lectin that binds to sulfated glycan ligands
doi: 10.1016/j.jbc.2026.111328
Figure Lengend Snippet: Activity of B4GALNT3 and its PA14-mutant toward glycoproteins. A , a His-tagged soluble form of B4GALNT3 (Y83) and its N252A mutant expressed in HEK293T cells were purified from the medium through a Ni 2+ -column and subjected to SDS-PAGE and CBB staining. B , transferrin and haptoglobin derived from human serum were pretreated with sialidase and galactosidase, incubated with purified B4GALNT3 WT, and blotted with anti-transferrin, anti-haptoglobin, and WFA. C , the similar experiment to ( B ) was performed using B4GALNT3 WT and N252A mutant. D , LH was treated with neuraminidase and galactosidase or with the same amount of vehicle and subjected to SDS-PAGE and CBB staining. E , LH and haptoglobin pretreated with neuraminidase and galactosidase were incubated with purified B4GALNT3 WT or N252A for 1 h or 3 h, and subjected to blotting with WFA ( left ), anti-haptoglobin ( right ), or CBB staining ( middle ).
Article Snippet: The following antibodies and lectins were used: mouse anti-GAPDH (Merck Millipore; MAB374), rabbit anti-B4GALNT3 (HPA011404), goat anti-transferrin (Cappel; 55,139), rabbit anti-haptoglobin (abcam; ab256454), mouse anti-Myc (Millipore; 05-724), mouse anti-chondroitin sulfate CS-56 (Seikagaku Corp; 270695), mouse anti-chondroitin sulfate LY111 (Tokyo Chemical Industry; A3143), mouse anti-VDAC1 (abcam; ab14734), HRP-anti-mouse IgG (GE Healthcare; NA931V), HRP-anti-goat IgG (Jackson ImmunoResearch; 705-035-147), HRP-anti-rabbit IgG (GE Healthcare; NA934V), Alexa488-anti-mouse IgM (Invitrogen; A21042), biotinylated WFA (Sigma; L1516).
Techniques: Activity Assay, Mutagenesis, Purification, SDS Page, Staining, Derivative Assay, Incubation
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