A) Cytosolic fractions were collected and analyzed by Western blot. Expression levels of the hexosamine biosynthetic pathway (HBP) proteins in cell lines representing prostate cancer (lane 1:PC3, lane 2:LNCaP and lane 3:VCaP) and benign prostate epithelial cells (lane 4:PNT2 and lane 5:RWPE-1). The intensity of the WB signal was determined by densitometry, normalized to actin and the amount in RWPE-1 cells was set to one. This experiment was repeated twice. LNCaP cells were treated either with a concentration gradient of OGT inhibitor, ST045849 (B) or an inhibitor of N-linked glycosylation, tunicamycin (C). Viability was assessed after 48 hours using an MTS assay and values were normalized to sample without any treatment. Assays were repeated three times, each time six technical replicates and SEM of the biological replicates is shown. Statistical analysis was performed with Student's t-test, (*<0.05, **<0.01, ***<0,001). D) LNCaP and VCaP cells were treated with tunicamycin (5 µg/ml) for the indicated time and protein lysates were harvested. The intensities of KLK3 and BiP signals were determined with densitometry, normalized to actin and the protein amount in normal condition was set to one. The relationship between the high and low mobility KLK3 is shown. This experiment was repeated twice.