Pugnac

Cells were lysed for 15 min at ice bath using a lysis buffer (1% Triton X-100, 20 mM Tris pH 7.5, 1 mM MgCl₂, 150 mM NaCl, 1 mM Na₃VO₄, 50 mM NaF, 1.5 mM EDTA, 10% glycerol, 20 mM β-glycerophosphate, 10 µg/ml aprotonin, 1 µM pepstatin A) containing 5 µM PUGNAc (an OGA inhibitor; Toronto Research Chemicals, Inc., North York, Canada). Protein samples (50 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany). The membrane was blocked with 5% non-fat dried milk in TBST for 1 h at room temperature and incubated overnight at 4°C with primary antibodies. Antibodies specific to O-GlcNAcylation (RL2; 1:1,000; Affinity BioReagents, Golden, CO, USA) and OGT (F-12; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used. RhoA (67B9; 1:1,000), MLC (3672; 1:1,000) and phosphorylated (p-)MLC (3671; 1:1,000) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). GAPDH antibody (sc-25778; 1:2,000) and horseradish peroxidase-linked goat anti-mouse (sc-2005; 1:2,000) and goat anti-rabbit (sc-2004; 1:2,000) IgG secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. Development was carried out using an enhanced chemiluminescence western blotting detection reagent (GE Healthcare Life Sciences, Chalfont, UK).

OSMI-1 (Sigma, 50 μM), 6-Ac-CAS (GlycoSyn, 10 μM), PUGNAc (Toronto Research Chemicals, 10 μM) were used when indicated. The Myc-hOGT plasmid was provided by Dr. Xiaochun Yu at the University of Michigan (Chen et al., 2013 (link)). Ngn3-luc was generated by inserting a ~4.1 kb promoter of the mouse Ngn3 gene into the pGL3-basic vector. IRE-luc was generated by inserting the −1985 to −280 bp upstream of the mouse Irs2 gene that contains two insulin-response elements (IREs) into the pGL3-basic vector. pCMV6-mNgn3-Myc-DDK (#MR212139) was purchased from Origene. pCMV-FOXO1 (#12148) was purchased from Addgene. pCMV-FOXO-4A was generated with the QuikChange XL II Site-Directed Mutagenesis Kit (Agilent). HepG2 and Caco-2 cells were transfected with expression plasmids, luciferase reporters, and β-galactosidase or Renilla-luciferase using Lipofectamine 3000 (Invitrogen) or FuGENE HD (Promega). Cells were lysed and luciferase and β-galactosidase enzyme activities were measured using kits from Promega. Relative luciferase activity was determined by normalizing to β-galactosidase or Renilla-luc activity (Ruan et al., 2012 (link); Wang et al., 2018 (link)).