Figure 6.

Abnormal intracellular PrP localization and impairment of VSV-G transport in human fibroblasts from carriers of the FFI mutation.A, to visualize PrP on the cell surface, human fibroblasts (HFs) from noncarriers (WT) and carriers of the PRNP D178N mutation (FFI) were immunostained with anti-PrP antibody 6H4 before fixation and application of Alexa Fluor 488 (green)-conjugated secondary antibody and staining with Hoechst 33258 (blue) to visualize the nuclei. To investigate the intracellular distribution of PrP, cells were fixed and permeabilized before immunostaining. B, FFI HFs were fixed, permeabilized, and immunostained with anti-PrP and anti-giantin antibodies, followed by Alexa Fluor-conjugated secondary antibodies. Cells were viewed with green excitation/emission settings to detect PrP, red excitation/emission settings to detect giantin, and UV excitation/emission settings to detect the nuclei. Scale bar 15 μm. C, HFs were infected with VSV at 32 °C for 1 h, washed, and cultured at 35 °C for 4 h. Cells were fixed and immunostained with monoclonal anti-VSV-G and polyclonal anti-GM130 antibodies, followed by Alexa Fluor-conjugated IgG secondary antibodies. Scale bar 10 μm. D, the normalized fluorescent density of VSV-G in the Golgi was measured and expressed as fold change over WT. Each bar indicates the mean ± SEM of 209 WT and 211 FFI cells (four independent lines each). ∗∗∗∗p < 0.0001 by unpaired t-test. Scale bar 10 μm.