Figure 8.

Visualization of Wld^S and variant ΔNLS Wld^S distribution in primary neuronal culture. A, Left, Western blot from SCG cell body/proximal neurite (“cell bodies”) and distal neurite fractions (“neurites”) showing Wld^S and variant Wld^S in neurites. Note the reduced level of variant ΔNLS Wld^S in cell bodies and significantly increased levels in neurites. To rule out nuclear contamination derived from glial and other cells in neurite fractions, Western blots were probed with the nuclear marker Histones H1. Right, Densitometric quantification of variant Wld^S protein (normalized to β-actin). Data from two independent experiments are presented as mean ± SD. B, Left, Confocal images showing cell bodies (and proximal neurites) from dissociated SCG preparations labeled with Wld18 antibody (green; Alexa488-tyramide signal amplification), neurofilament antibody (red) and DAPI (blue). Note cytoplasmic redistribution of the ΔNLS Wld^S protein variant relative to Wld^S. Right, Higher magnification confocal images demonstrating variant Wld^S in SCG neurites (green; Alexa488-tyramide signal amplification). C, D, High-power confocal projections demonstrating peri-nuclear variant Wld^S foci (green) in SCG preparation from ΔNLS Wld^S transgenic mouse (C) and occasional foci in proximal neurites (D, arrows). E, Confocal projection showing transfected hippocampal neuron expressing ΔNLS Wld^S-EGFP fusion protein. Note cytoplasmic ΔNLS Wld^S-EGFP foci in cell body and neurites.