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. 2013 Jul;183(1):247–256. doi: 10.1016/j.ajpath.2013.04.001

Figure 4.

Figure 4

Mutant decorin lacks O-linked GAG chains. A: HEK293T cells were transfected with vector only, vector carrying WT decorin cDNA, or vector carrying mutant decorin cDNA. Decorin mRNA was undetectable in cells transfected with vector only. mRNA levels of decorin were comparable between cells transfected with either WT or mutant decorin, indicating similar transfection efficiency between the two groups. B: At 60 hours after transfection, proteins were collected from the cells. There was virtually no endogenous decorin expression in HEK293 cells transfected with vector only. Substantial decorin expression was observed in cells transfected with either WT or 952delT decorin. The samples were digested sequentially with ChABC and PNGase F. WT decorin bands became sharper and thicker after removing the O-linked GAG chain with ChABC; however, there was no change in the 952delT decorin band, indicating lack of a GAG chain in the 952delT mutant decorin. Furthermore, N-deglycosylation by PNGase F induced a shift in all of the O-deglycosylated bands on the immunoblots. The distance between the WT and truncated decorin bands after O-deglycosylation remained constant on the immunoblots after sequential deglycosylation with ChABC and PNGase F.