Figure 4

taken from 1-day-old adult flies. (d) Western analysis of phospho-tau protein (AT8) levels from 1-day-old fly heads of the indicated tau isoforms under the control of GAD-Gal4. The blots are stripped and re-probed with α-Tubulin, then stripped and re-probed with total tau to serve as loading controls. The images are cropped from the same blot, and the full-length blot refers to figure S11. (e) Confocal images of immunolabeling of anti-hTau (red) from the brains of 202508-Gal4 driving lacZ and the indicated tau isoforms with the co-expression of syt-GFP. Images are taken from 10-day-old adult flies. Scale bars: 10 μm (a, upper panel), 20 μm (a, lower panels), 20 μm (b), 10 μm (c, e). (f) Quantification of Syt-GFP and tau colocalization, as shown in (d). Six brains are measured for each genotype. See “Methods” for the detail of Pearson correlation analysis. All the examined flies were 10-day-old (n = 6, ***p < 0.001, one-way ANOVA with Bonferroni multiple comparison tests). (g) Western blot analysis of GABA neurons expressing lacZ (control) and the indicated tau isoforms with a GAD-Gal4 driver. Different fractions of neuronal extracts, including nucleus/high-density cellular components and synaptosomes, are separated for analysis. Syntaxin is a marker of synaptosome, and histone H3 is a marker of nucleus. The blots are stripped and re-probed with syntaxin and histone H3 sequentially to serve as loading controls. The images are cropped from the same blot, and the full-length blot refers to Figure S12. (h) Quantification of replicated results shown in (f) the relative ratio of tau in each fraction is calculated as following: R1 = nucleus tau/histone H3, R2 = synaptosome tau/syntaxin; tau% in the nucleus = R1/(R1 + R2), tau% in the synaptosome = R2/(R1 + R2), **p < 0.01 (n = 5, two-way ANOVA with Bonferroni multiple comparison tests).