Anti oga

Forty micrograms of cell proteins were separated by SDS electrophoresis under denaturating conditions using 6–10% polyacrylamide gels. SDS-PAGE gels were electrophoretically transferred on PVDF membrane in Tris-glicine buffer, using the Mini Transblot System (Bio-Rad Laboratories, Richmond, VA). O-GlcNAc levels were measured by anti-b-O-linked N-Acetylglucosamine (OGlcNAc) CTD 110.6, an antibody that specifically recognizes endogenous levels of O-GlcNAc, linked to both serine and threonine residues of proteins, 1:1000 dilution (Cell Signaling). Other primary antibodies were used as follows: anti-OGA 1:3000 dilution (Sigma-Aldrich), anti-OGT 1:500 dilution (Sigma–Aldrich), and anti-Histone H3 1:2000 dilution (Cell Signaling).
Each membrane was washed three times for 10 min and then incubated with the appropriate secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) for 1 h. For the immunological detection of proteins, MINI HD 9 System (Uvitec Limited, Cambridge UK) was used. Band density was quantified Quantity One Software (Bio-Rad Laboratories).

PVAT from thoracic aorta and visceral adipose tissue from humans were frozen in liquid nitrogen and homogenized in a lysis buffer [50 mM Tris/HCl, 150 mM NaCl, 1% Nonidet P40, 1 mM EDTA, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM sodium fluoride]. The tissue extracts were centrifuged, and total protein content was quantified using the Bradford method (Bradford, 1976 (link)). Proteins (20 μg) were separated by electrophoresis on 10% polyacrylamide gel, and transferred on to nitrocellulose membranes. Non-specific binding sites were blocked with 5% bovine serum albumin (BSA) in Tris buffered saline (TBS) containing 0.1% Tween 20 (for 1 h at 24°C). Membranes were incubated with antibodies (at the indicated dilutions) overnight at 4°C. Antibodies were used as follows: anti-Anti-β-O-Linked N-Acetylglucosamine (1:5000 dilution; Sigma-Aldrich Inc., Germany), anti-OGA (1:1000 dilution; Sigma-Aldrich Inc., Germany), anti-OGT (1:1000 dilution; Abcam, UK), anti-GAPDH (1:10000 dilution; Sigma-Aldrich Inc., Germany). After incubation with secondary antibodies, signals were obtained by chemiluminescence and quantified densitometrically.