Confluent cultured cells were preincubated with or without 2.5 mM alloxan (an inhibitor of OGT that reduces O-GlcNAc protein levels)
26 for 1 hour, 100 μM O-(2-acetamido-2-deoxy-
d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc, an inhibitor of N-acetylglucosaminidase [O-GlcNAcase, OGA] that increases O-GlcNAc protein levels)
27,28 for 1 hour, 2.5 μM MG-132 for 1 hour, or 2 μg/mL tunicamycin for 18 hours. They were then further incubated with or without 5 mM GlcN for 1 hour before stimulation with TNF-α (20 ng/mL) for 24 hours at 37°C. To measure ICAM-1 and O-GlcNAc, the cells were washed twice with PBS and detached by scraping. The cells were pelleted at 1000
g, resuspended, and sonicated in cold lysis buffer (50 mM Tris-HCl [pH 7.5] 2% SDS, and 1 mM phenylmethylsulfonyl fluoride). The insoluble debris was removed by centrifugation at 16,000
g at 4°C for 10 minutes. The protein content was determined using the bicinchoninic acid method (Pierce, Rockford, IL) with BSA as the standard. The lysates (20 μg) were resolved with one-dimensional SDS-PAGE (10% polyacrylamide gels). The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA) that had been blocked with 5% (wt/vol) milk for 1 hour at room temperature, and then incubated overnight at 4°C with antibodies directed against ICAM-1 (diluted 1:1000 in Tris-buffered saline containing Tween-20 (TBST, 0.1% at 1X); Santa Cruz Biotechnology, Santa Cruz, CA), O-GlcNAc (CTD110.6; diluted 1:2000 in TBST; Covance, Berkeley, CA), OGT (diluted 1:1000 in TBST; Santa Cruz Biotechnology), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; diluted 1:25,000 in TBST; Santa Cruz Biotechnology), and Na
+/K
+ ATPase (diluted 1:1000 in TBST; Santa Cruz Biotechnology). The membranes were washed and incubated with a horseradish-peroxidase-conjugated secondary antibody (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature, and the protein was visualized with an enhanced chemiluminescence procedure (enhanced chemiluminescence reagent; Millipore, Billerica, MA). The mean protein levels were measured densitometrically with Image J 1.46a (National Institutes of Health, Bethesda, MD).