In Vitro O-GlcNAcylation Assay

Each reaction contained 1 μg recombinant lamin tails, 1 Unit calf intestinal phosphatase (CIP) (New England Biolabs, Ipswich, MA, USA) and 10 mM UDP-GlcNAc (Sigma-Aldrich), plus or minus 1 μg purified recombinant active His-tagged OGT enzyme in a final reaction volume of 20 μL in 50 mM Tris-HCl pH 7.4. Recombinant OGT was purified as described [32 (link)]. Reactions were incubated for 2 h at 22–25 °C, and then overnight at 4 °C. Reactions were stopped by adding 4X SDS sample buffer and 33% of each reaction was resolved on 4–12% Bis-Tris NuPage gels (Invitrogen), and then transferred to nitrocellulose membranes, blocked for 1 h at 22–25 °C with 3% BSA in PBS, and incubated overnight at 4 °C with affinity-purified O-GlcNAc-specific mouse antibody CTD_110.6 (diluted 1:1000), or with a mixture of CTD_110.6 antibody and competing (free) GlcNAc sugar (100 mM). Secondary antibodies were horseradish peroxidase-coupled anti-mouse IgM (Santa Cruz SC-2064; diluted 1:10,000) and anti-T7-tag (69048-3; diluted 1:100,000, Novagen, Darmstadt, Germany).