Horse anti mouse

The primary and secondary antibodies used in the present study were as follows: Ras-related GTP-binding protein D (RRAGD; cat. no. A304-301A-T; Thermo Fisher Scientific, Inc.), GAPDH (cat. no. A300-642A-T; Thermo Fisher Scientific, Inc.), goat anti-rabbit (cat. no. 7074; Cell Signaling Technology, Inc.) and horse anti-mouse (cat. no. 7076; Cell Signaling Technology, Inc.).
The reagents used were as follows: FBS, DMEM, penicillin-streptomycin, Lipofectamine 2000^®, TRIzol^® reagent (all from Thermo Fisher Scientific, Inc.); the PrimeScript qRT Reagent kit (Takara Bio, Inc.), SYBR-Green Mix (Roche Diagnostics), the Dual Luciferase Assay System (Promega Corporation), protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA), RIPA buffer (Roche Diagnostics), chemical HRP substrate (MilliporeSigma), the Annexin-V/Dead Cell Apoptosis kit, glucose uptake colorimetric assay kit (BioVision, Inc.), 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Sigma-Aldrich; Merck KGaA), Krebs-ringer-phosphate-HEPES (KRPH; Thermo Fisher Scientific, Inc.), the lactate colorimetric assay kit (BioVision, Inc.) and the XF Glycolysis Stress Test kit (Seahorse Bioscience).

Proteins were extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Subsequently, the proteins (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with the corresponding primary antibodies: Anti-B3GNT5 (1:1,000; cat. no. PA5-26653; Thermo Fisher Scientific, Inc.) and anti-GAPDH (1:5,000; cat. no. ab8245; Abcam) at 4°C overnight. After washing the membrane with Tris-buffered saline with 0.1% Tween-20, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies: Goat anti-rabbit secondary antibody (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) and horse anti-mouse (1:3,000; cat. no. 7076; Cell Signaling Technology, Inc.) at room temperature for 1 h. Protein bands were imaged using an Enhanced Chemiluminescence Kit (Beyotime Institute of Biotechnology). The intensity of the protein bands was analyzed using ImageJ 1.51 software (National Institutes of Health).