Ab96718

Antibodies against OGT (ab96718), OGA (MGEA5, ab124807), O-GlcNAc (RL2, ab2739), PLIN1 (ab3526), SNAP23 (ab3340), ATGL (ab99532), DGAT1 (11561-1-AP), Fsp27 (CIDE C, ab77115), and p-Ser (phosphoserine, PSR-45) (Abcam); p492-phosphorylated PLIN1 (4855) and p517-phosphorylated PLIN1 (4856) (Vala Sciences); HA (H3663) (Sigma-Aldrich); CGI-58 (ABHD5, 12201-1-AP), PLIN2 (15294-1-AP), and PLIN3 (TIP47, 10694-1-AP) (Proteintech); p563-HSL (4139), phospho-Akt (Ser473, 9271), and Akt (9272) (Cell Signaling Technology); Myc (sc-40), DGAT2 (sc-66859), and β-actin (sc-8432) (Santa Cruz Biotechnology) were purchased from the indicated sources. Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Alexa Fluor 594-conjugated secondary antibodies, BODIPY 493/503, and BODIPY 558/568 C12 fatty acid were obtained from Thermo Fisher Scientific. 4′,6-Diamidino-2-phenylindole (DAPI), OA, Fsk, and IBMX were from Sigma-Aldrich. CL-316,243 was from R&D Systems. OGT inhibitor ST045849 (ST04) was purchased from TimTec. TMG was from Cayman Chemical.

Paraffin-embedded sections of thoracic aorta were permeabilized and dehydrated, followed by treatment with 3% H₂O₂ to block endogenous peroxidase activity. Antigen retrieval was performed using citric acid buffer. After being blocked by normal goat serum (Sangon Biotech Biotechnology Co., Ltd., Shanghai, China), the sections were incubated with primary antibody of rabbit anti-human/rat OGT (ab96718, dilution ratio of 1:500, Abcam) and rabbit anti-human/rat KEAP1 (ab256813, dilution ratio of 1:500, Abcam) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG; ab6721, dilution ratio of 1:5000, Abcam) secondary antibody for 30 min at 37°C. After color development with a diaminobenzene kit (Sigma, United States), the sections were subjected to hematoxylin staining, dehydration, permeabilization, and neutral gum mounting, and followed by observation and photographing under an upright microscope (BX63, Olympus, Japan).

Antibodies against YTHDF2 (ab220163), O-GlcNAc (ab2739), OGA (ab124807), OGT (ab96718), K48 (ab271911) and p-Rb (ab173289) were obtained from Abcam (Cambridge, UK). Antibodies against MCM2 (10513-1-AP), MCM5 (11703-1-AP), and β-Tubulin (66240-1-Ig) were obtained from Proteintech (Rosemont, IL, USA). Antibody against HA (26183) was from Invitrogen (Carlsbad, CA, USA). Antibody against FLAG (F3165) was from Sigma-Aldrich (Germany). Antibody against H3 (H0164) was from Millipore (Massachusetts, USA). Antibodies against Rb (A16966), p21 (A21749), cyclinD1 (A2708) and CDK4 (A0366) were from ABclonal Technology (Wuhan, China). Antibodies against β-actin (TA-09), Goat rabbit IgG/TRITC, secondary (ZF-0316), and Goat mouse IgG/FITC, secondary (ZF-0312) were purchased from ZSGB-BIO (Beijing, China). Antibody against OGT (sc-74546, for immunofluorescence) was from Santa Cruz (CA, USA).

Jejunal IECs were lysed in a RIPA buffer containing proteinase inhibitors and an OGA inhibitor. Protein extracts were separated on an SDS-PAGE gel, transferred to nitrocellulose membranes, and blotted with the indicated primary antibodies: OGT (ab96718, Abcam, Shanghai, China), O-GlcNAc (ab2739, Abcam), α-Tubulin (11224-1-AP, Proteintech, Wuhan, China), P-STAT6 (Y641) (ab263947, Abcam), and STAT6 (51073-1-AP, Proteintech). After incubation with HRP-conjugated secondary antibodies, the immune complexes were detected using the ECL detection reagents (Beyotime, Shanghai, China).

Mice were transcardially perfused with saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS), pH 7.4. The brains were removed, postfixed overnight in 4% PFA at 4°C, transferred to 30% sucrose in 0.1 M PBS, pH 7.4, and coronally sectioned (40 μm) on a cryostat (Leica CM3000). After washing three times with PBS, the sections were incubated in blocking buffer containing 5% normal goat serum in 0.2% Triton X-100/PBS (PBST) for 1 h at room temperature. Then, with primary antibodies against OGT (Abcam, ab96718, 1:250, RRID: AB_10680015), glial fibrillary acidic protein (GFAP) (Cell Signaling Technology, 3670, 1:300, RRID: AB_561049), and NeuN (Millipore, MAB377, 1:500, RRID: AB_2298772) in blocking buffer overnight at 4°C. After washing, the samples were incubated with secondary antibody (Invitrogen, A11032, 11034, 11037, 11077, RRID: AB_2576217, AB_2534095, AB_2534091, and AB_2534121) for 1 h at room temperature. Nuclei were counterstained with DAPI. Samples were mounted on glass slides and then cover slipped with anti-fade solution, visualized using a Nikon fluorescence microscope. Mean intensity levels of GFAP were measured by ImageJ.

NCI-H1299 cells were lysed in SDS lysis buffer (1% SDS, 50 mM Tris-HCl pH 7.5, 100 mM NaCl, and Complete™ Protease Inhibitor). Lysates were resolved on 4-12% SDS polyacrylamide gels (SDS PAGE), transferred to Immobilon-FL PVDF membranes (#IPVH00010, MerckMillipore), and immunoblotted with the indicated antibodies. Blots are identified with the antibody, dilution and clone/catalogue number in parentheses. The antibodies used were anti-OGT (1:1000, ab96718, Abcam), Goat Anti-Rabbit IgG H&L (HRP) (1:2000, ab6721, Abcam), and HRP-Streptavidin (1:5000, RABHRP3, Sigma-Aldrich).

HeLa cells were co-transfected with HA-PRK5-USP7 and pCDEF3-MLL5-FLAG and pCDNA3-OGT-V5 plasmids using lipofectamine 2000 (Invitrogen). 48h after transfection, cells were fixed for 1h with 2% PFA and permeabilized with 1% Triton-X-100. Cells were then blocked with blocking buffer (0.5% Triton X-100, 3% BSA, 10% NCS in PBS) for 1h at room temperature. Cells were stained with monoclonal anti-FLAG (F1804, Sigma), monoclonal anti-HA (11867423001, Roche) and polyclonal anti-OGT (Ab96718, Abcam) antibodies. Donkey-anti-mouse Alexa Fluor-555 (A21422, Invitrogen), Donkey-anti-rabbti Alexa Fluor-488 (A110081, Invitrogen) and Donkey-anti-Rat Alexa Fluor-633 (A21082, Invitrogen) were used as secondary antibodies. Cell nuclei were stained with DAPI. The slides were imaged with a laser confocal microscope (Leica SP5).