Figure 2.

Reduced nuclear targeting of ΔNLS Wld^S in various neuronal subtypes in vivo. A, Western blots of nuclear, cytoplasmic, and cytosolic brain fractions demonstrating dramatically reduced nuclear targeting in individual mice from different ΔNLS Wld^S lines compared with spontaneous Wld^S mutant. The bottom graph demonstrates corresponding relative optic densities of nuclear Wld^S bands normalized to SP1 for individual mice. These Western blots are representative of three individual experiments. B, Rows 1–6, Confocal images showing (variant) Wld^S protein immunolabeling (red) with β-III tubulin (green) and Hoechst 33258 (blue) counterstaining on frozen sections from homozygous mice. Transgenics shown represent lines without detectable variant Wld^S protein expression in Western blotting (line 1) and with strong (line 2) and medium (line 3) variant Wld^S protein expression levels. Row 7, Light microscopic pictures from lumbar motorneurons (arrows) showing Wld^S immunolabeling using 3,3′-diaminobenzidine. C, Graph showing normalized fluorescence intensities for nuclear variant Wld^S protein signals in lumbar motoneurons. For each genotype group, three mice were assessed (10 motorneurons each). *p < 0.05; **p < 0.005; ***p < 0.001; one-way ANOVA.