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. 2021 Mar 1;296:100490. doi: 10.1016/j.jbc.2021.100490

Figure 4.

Figure 4

Retention of nonaggregated PrP in the ER or Golgi does not impair VSV-G trafficking.A, HeLa cells were transfected with plasmids encoding WT, PrP-ER, PrP-Golgi, or PG14 PrP. After 24 h cells were lysed in nondenaturing detergents, and the cell lysates were ultracentrifuged at 186,000g for 45 min. PrP in the supernatant (S) and pellet (P) was analyzed by western blot. Molecular weight markers are in kDa. B, HeLa cells were transfected with plasmids encoding WT, PrP-ER, PrP-Golgi or PG14 PrP, and VSV-G-EGFP fusion protein. After 24 h at 35 °C cells were fixed, permeabilized, stained with monoclonal anti-PrP 12B2 antibody followed by Alexa Fluor-conjugated anti-IgG secondary antibody. Cells were viewed with green excitation/emission settings to detect VSV-G and redexcitation/emission settings to detect PrP. Scale bar 10 μm. C, the normalized fluorescent density of VSV-G at the plasma membrane (PM) was measured in PrP-expressing cells and expressed as the fold difference from WT PrP-transfected cells. Each bar indicates the mean ± SD of 9 to 21 cells; ∗∗∗∗p < 0.0001 versus WT by one-way ANOVA, Tukey’s post-hoc test.