(A) Brain lysates of 2 and 10 days after eclosion (DAE) wild-type (WT) flies (lanes 1 and 6), flies expressing human Tau with GFP (lanes 4 and 9), or human Tau with Hsp27 (lanes 5 and 10) in the nervous system were probed with antibodies for disease-associated phospho-tau epitopes S262, Ser202/Thr205 (AT8), and total Tau (5A6). Actin was probed as a loading control. Brain lysates of flies carrying only UAS elements were loaded for control (lanes 2, 3, 7, and 8). (B) Quantification of protein fold changes in (A). The levels of Tau species were normalized to actin. Fold changes were normalized to the Tau +GFP group at 2 DAE. n=3. (C) Brains of WT flies or flies expressing Tau +GFP or Tau +Hsp27 in the nervous system at 2 DAE were probed for AT8 (heatmap) and Hsp27 (green), and stained with DAPI (blue). Scale bar, 30 μm. (D–F) Quantification of the Hsp27 intensity (D, data normalized to WT), brain optic lobe size (E), and AT8 intensity (F, data normalized to the Tau +GFP group). n=4. (G) Brain of flies expressing Tau +GFP or Tau +Hsp27 in photoreceptors were probed for AT8, Hsp27, bruchpilot (BRP), and horseradish peroxidase (HRP). Scale bar, 30 μm. (H, I) Quantification of AT8 intensity (H) and BRP intensity (I). Data were normalized to the Tau +GFP group. n=5. Statistical analyses were performed using one-way ANOVA with Bonferroni’s post hoc test (B, D, E) or independent samples t-test (F, H, I). All data are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.