Anti o glcnac

For protein extraction, the mycelia of different strains cultured in liquid CM for 48 h were collected, which were ground into powder in liquid nitrogen and resuspended in protein extraction buffer (Biyuntian, Beijing, China). To detect O-GlcNAcylation level, the total protein was immunoprecipitated using anti-Flag beads (Abmart, Shanghai, China), which was then separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). The PVDF membrane was then incubated with anti-O-GlcNAc as the primary antibody (1:5000, Sigma-Aldrich, St. Louis, MO, USA) and anti-rabbit horseradish peroxidase as the secondary antibody (1:10,000, Abmart, Shanghai, China). To detect protein level of Def1, the total protein was separated on a 10% SDS-PAGE gel and, then, transferred onto a PVDF membrane, which was incubated with anti-Flag as the primary antibody (1:5000, Abmart, Shanghai, China) and anti-rabbit horseradish peroxidase as the secondary antibody (1:10,000, Abmart, Shanghai, China).

Cells were collected and washed three-times with ice-cold PBS to remove residue media. Then cell pellets were resuspended in cold RIPA lysis buffer (150mM NaCl, 1% NP40, 0.5% DOC, 50mM Tris-HCl at pH 8, 0.1% SDS, 10% glycerol, 5mM EDTA, 20mM NaF and 1mM Na3VO4), supplemented with protease inhibitors. Cell debris was removed by centrifugation at 15000 rpm for 20 minutes at 4°C using benchtop centrifuge. Protein concentration was determined flowing Bradford assay. 50 µg of protein from each sample was separated by SDS-PAGE and transferred to PVDF membrane. Target proteins were detected using indicated specific antibodies: Anti-OGT (Cell-Signaling, cat #20438), Anti-O-GlcNAc (Sigma, cat #MABS1254), Anti-c-MYC (Novus, cat #NB600-335), Anti-Actin (Santa Cruz, cat #sc-47778), Anti-NANOG (Cell-Signaling, cat #3580), Anti-SOX2 (Cell-Signaling, cat #3579), Anti-OCT4 (Cell-Signaling, cat #2750), Anti-KLF8 (Sigma, cat #AV32859).

HCT116 and HepG2 cell samples were lysed with NP40 assay buffer containing 1× PBS, 1% NP40, 1 mM EDTA, and protease inhibitor cocktail tablets (Roche, Mannheim, Germany). Proteins were separated on 8–15% SDS-PAGE gels and transferred to nitrocellulose membranes (GE Healthcare Life science, Boston, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight with primary antibodies at 4 °C. Anti-O-GlcNAc was purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-PARP was purchased from Invitrogen (Waltham, MA, USA). Anti-total caspase-3, cleaved caspase-3, IRE1α, p-p65, p65, p-IKKα/β, IKKα/β, p-IκB and p-eIF2α were purchased from Cell Signaling Technology. Anti-OGT, PERK, Bcl2, Bax, cytochrom c and β-actin were purchased from Santa Cruz. Membranes were washed with TBST and incubated with antimouse or antirabbit horseradish peroxidase (HRP)-conjugated IgG secondary antibody for 30 min. The signal was visualized using an ECL Plus Detection System (GE Healthcare Life Sciences) and the bands were quantified using ImageJ software (NIH, United States).

DMEM (Invitrogen, Carlsbad, CA, USA), fetal bovine serum (Gibco, USA), and anti-eNOS (Cat No. 32027, Cell Signaling Technology, USA), anti-AMPK (Cat No. 9158, Cell Signaling Technology, USA), anti-O-GlcNAc (Cat No. 05-1245, Sigma, USA), anti-O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) (Cat No. ab177941, Abcam, England) anti-protein O-GlcNAcase (OGA) (Cat No. ab124807, Abcam, England), goat anti-mouse IgG (Cat No. SA00001-1, Proteintech, USA), and goat anti-rabbit IgG (Cat No. SA00001-2, Proteintech, USA) antibodies were used. Other reagents are mentioned as they appear in the article.