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. 2011 Sep 19;286(47):40962–40973. doi: 10.1074/jbc.M111.283978

FIGURE 3.

FIGURE 3.

Swainsonine treatment of PK1, LD9, CAD5, and R33 cells renders glycans susceptible to Endo-H cleavage. Cells were propagated in 2 μg of swa/ml for the times indicated, and cell lysates, digested or not with Endo-H or PNGaseF, were analyzed by Western blotting. Misglycosylation of PrP was evident after 1 day of swa treatment, as shown by increased electrophoretic mobility of the slowest PrPC band (diglycosylated PrPC) for all cell lines. The PrP-linked glycans of PK1 and LD9 became fully susceptible to Endo-H treatment after 1 day of swa treatment, but in the case of CAD5 and R33 cells some PrP remained Endo-H-resistant even after 4 days. “++” designates 4-day samples that were treated twice with Endo-H to ensure complete cleavage of all susceptible glycans. The arrows point to residual endo H-resistant PrPC. C2 is a fragment arising from cleavage between positions 110/111 of PrPC in uninfected cells (62). C1, due to cleavage around position 90, is usually predominant in prion-infected cells but has also been reported in uninfected cells (63). The data shown are from one of three experiments.