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. 2023 Jul 27;299(9):105101. doi: 10.1016/j.jbc.2023.105101

Figure 6.

Figure 6

Expression of PrP constructs in N2a cells.AC, N2a cells in which endogenous PrP expression was eliminated by gene editing were transfected with plasmids encoding WT PrP (A) or N180Q/N196Q PrP (B), along with an EGFP plasmid as a transfection marker. Cells in (C) were transfected with the EGFP plasmid alone. Cells were then fixed without permeabilization and immunostained for PrP using D18 antibody. Images show fluorescence for PrP, EGFP, DAPI, and a merge of all three channels. The results show that N180Q/N196Q PrP is present on the cell surface, similar to WT PrP. The scale bar represents 10 μm. D, quantification of cell surface expression of PrP on N2a cells transfected with WT and N180Q/N196Q PrP plasmids. Cell-surface fluorescence intensity for PrP was assessed in A and B using ImageJ software. Staining for PrP in C was undetectable. Bars show mean ± SE; n, number of cells used for quantitation. Cell surface expression of PrP was not significantly different between the two groups (ns). E, Western blot analysis of N2a PrP knockout cells transfected with vector alone (KO) or with plasmids encoding WT, ΔCR, H139Y/H176Y, N180Q/N196Q, and H139Y/H176Y/N180Q/N196Q PrP. The latter two constructs, which carry mutation of both glycosylation sites (N180Q and N196Q), show an unglycosylated band (arrow), as well as lower Mr bands corresponding to cleavage products.

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