O glcnac

O-GlcNAc (1:1000, #12938S), OGT (1:1000, #23177S), AKT (1:1000, #90272), phospho-AKT (Ser473) (1:1000, #12694), PFKFB2 (1:1000, #13045), phospho-PFKFB2 (Ser483) (1:1000, #13064), phospho-GSK3β (Ser9) (1:1000, #9323), AS160 (1:1000, #2670), phospho-mTOR (Ser2448) (1:1000, #5536), and LC3A/B (1:1000, #12741) antibodies were purchased from CST (Cell Signaling Technology, Danvers, MA, USA). Anti-GSK3 beta (1:8000, #ab32391) and Anti-TBC1D4 (phosphor T642) (1:5000, # ab131214) antibodies were purchased from Abcam (Amyjet Scientific Inc., Upper Heyford, UK). Bax (1:2000, #60267-1-lg), Bcl-2 (1:2000, #60178-1-lg), caspase-3 (1:500, #66470-2), and mTOR (1:1000, #20657-1-AP) antibodies were purchased from Proteintech (Rosemont, IL, USA). Secondary antibodies were labeled with horseradish peroxidase goat anti-mouse IgG (1:20,000, # SA00001-1, Proteintech, USA), goat anti-rabbit IgG (1:20,000, #SA00001-2, Proteintech, USA), and goat anti-mouse IgG, IgM, IgA (1:10,000, ThermoFisher, Munich, Germany, #A-10668).

Western blot assay was carried out referring to a previous publication [24 (link)]. Antibodies, including AMPKα (cat. no.2532), phosphor-AMPKα (Thr172) (cat. no. 2531), ACC (cat. no.3662), phosphor-ACC (Ser79) (cat. no.3661), and O-GlcNAc (cat. no.9875), were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (cat. no. 309154) and HRP-conjugated secondary antibodies were purchased from Golden Bridge Biotechnology (Beijing, China).

Antibodies that recognize GFAT1 (ab125069, 1:1000), p38α (ab59461, 1:1000), TTLL5 (ab187697, 1:1000), TAB1 (ab76412, 1:1000) and β-actin (ab8227, 1:1000) were purchased from Abcam. Antibodies that recognize p62 (sc-28359, 1:1000), O-GlcNAc (sc-59623, 1:1000) LC3B (sc-271625, 1:1000) and p-p38 (sc-7973, 1:1000) were obtained from Santa Cruz. Antibody against Flag (F1804, 1:1000) was purchased from Sigma. Antibodies against p-MAPKAPK-2 (#3044, 1:1000), p-JunB (#8053, 1:1000), p-Acc (#11818, 1:1000), AMPK (#2603, 1:1000), JNK (#9252, 1:1000), p38α (#9218, 1:1000), p-AMPK (#50081, 1:1000), p-JNK (#4668, 1:1000), p-MKK3/6 (#9231, 1:1000), LC3B (#3868, 1:1000), p-p38 (#4511, 1:1000), His (#9991, 1:5000), GST (#5475, 1:5000) and O-GlcNAc (#9875, 1:1000) were purchased from Cell Signaling Technology. Antibodies that recognize TAB1 (D122651, 1:1000), HA (D191044, 1:1000) were purchased from Sangon Biotech. Antibody GT335 was purchased from Adipogen. Antibodies that recognize p38α (#48644, 1:1000) and β-actin (#49294, 1:1000) were purchased from Signalway Antibody. Phospho Ser438 TAB1 antibody (AF8324, 1:1000) was purchased from Affinity. GFAT1 antibody (14132-1-AP, 1:1000) and Caspase 3 antibody (19677-1-AP) were purchased from Proteintech.

Cultured HUVECs were collected and lysed with lysis buffer containing 98% RIPA lysis buffer, 1% protease inhibitor, and 1% phosphatase inhibitor. The cells were incubated with the lysis buffer on ice for 30 min. The total protein concentration in the lysate was measured using the BCA protein assay kit from Beyotime Biotechnology, Shanghai, China. Equal amounts of protein from each sample were loaded onto SDS-PAGE gels and separated by electrophoresis. The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with PBST containing 5% skim milk blocking buffer at room temperature for 2 h.
Primary antibodies, including O-GlcNAc (Cell Signaling Technology, #9875), GSDMD (Santa Cruz Biotechnology, sc-393581), GSDMD (Abcam, ab209845), β-actin (SAB, #21338), GAPDH (Proteintech), OGT (Santa Cruz Biotechnology, sc-74546), Myc Tag (Invitrogen, # R951-25), OGA (Proteintech, 14711-1-AP), were incubated with the membranes followed by incubation with HRP-conjugated secondary antibodies (anti-rabbit IgG from Bioword, and anti-mouse IgG from Bioword). Protein bands were detected using an Enhanced Chemiluminescent (ECL) reagent from Thermo Scientific, and the images were acquired with a ChemiDoc MP System from Bio-Rad. Densitometric analysis was performed using ImageJ Software to quantify the protein bands.