O glcnac

All primary and secondary antibodies used for immunoblotting were used at a concentration of 1:1000 and 1:10,000 dilution accordingly. Antibodies used in these studies were as follows: O-GlcNAc (RL2, ThermoFisher #MA1-072), actin (Sigma Catalog # A5316), α-Tubulin (sigma Catalog # T5168), OGT (AL-34) and OGA (345) antibodies were a generous gift from the laboratory of Dr. Gerald Hart (Department of Biological Chemistry, University of Georgia), Phospho-ERK (CST Catalog #9101), Total-ERK (Santa Cruz, Catalog # sc-93), phospho-MEK (ThermoFisher Catalog # 44-454G), Total MEK (ThermoFisher Catalog # 13-3500), APP (CST Catalog # 2452), and DUSP4 (CST Catalog # 5149).

Cells were cultured in six‐well plates with SBSGL at 0, 600, 1200 and 1800 μg/mL concentrations. Radio immunoprecipitation assay buffer (Shanghai Biyuntian Biotechnology Co., lot no. P0013C) with a protease inhibitor was used to extract proteins. Equal amounts of protein from each sample were transferred to polyvinylidene fluoride membranes (Merck, lot no. IPFL00010). Membranes were incubated with primary antibodies against RACK1 (Cell Signaling Technology, lot no. 5432, 1:1000), OGT (Cell Signaling Technology, lot no. 24083, 1:1000), O‐GlcNAc (Thermo Fisher Scientific, lot no. MA1‐072, 1:1000), LC3B (Cell Signaling Technology, lot no. 3868, 1:1000), p62 (Cell Signaling Technology, lot no. 5114, 1:1000), PD‐L1 (Cell Signaling Technology, lot no. 13684, 1:1000), Myc‐tag (Cell Signaling Technology, lot no. 2276, 1:1000) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, Cell Signaling Technology, lot no. 5174, 1:1000) overnight at 4°C. After washing with Tris‐buffered saline with Tween (TBST, Biosharp, lot no. 21259419), secondary antibodies goat anti‐rabbit IgG‐HRP (Abbkine, lot no. A21020, 1:10,000) and goat anti‐mouse IgG‐HRP (Abbkine, lot no. A21010, 1:10,000) were applied for 1 h at room temperature. Membranes were washed thrice with TBST, and visualization was achieved using an ECL luminescence kit (Shanghai Biyuntian Biotechnology Co., lot no. P0018S).

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).