|
Loss of solubility, truncation, and oligomerization of Human 4R/0N-Tau in transgenic Tau zebrafish. Proteins were extracted from Ctrl or Tau zebrafish pooled head regions, using RIPA (SDS/Triton), DIGE (7 M urea) or TBS (tris-buffered saline) for analysis by western blot. a Comparison of Ctrl and Tau zebrafish with postmortem midbrain samples from 3 different PSP patients. Blots probed with antibodies to total human Tau (above) and β-Actin (below). b Analysis of Tau zebrafish with increasing age. Blots were probed with antibodies to total human Tau (above), phosphorylated human Tau (AT8; middle) and β-Actin (below). c Real-time quantitative RT-PCR quantification of Hsa.MAPT mRNA in Tau zebrafish at 3–6 dpf. Each data point shows a biological replicate (calculated as mean of three technical replicates). Bars show group mean ± SE; ns not significant, 1-way ANOVA with Dunnett’s multiple comparisons test. d Western blot similar to b but run to resolve lower molecular weight proteins and probed with an antibody to human pS422-Tau. e Western blot of Tau and Ctrl zebrafish samples at 5dpf probed with antibody TauC3 that specifically recognizes human Tau truncated by Caspase-3 at residue D421. *Denotes bands with sizes suggestive of 0N/4R-Tau cleavage in Tau zebrafish. f Confocal micrographs showing sections from Tau and Ctrl brain labeled with the same TauC3 antibody used in e recognizing human Tau truncated at D421 (TauC3; green) and a nuclear counter label (DAPI; blue). Scattered labeled neurons in Tau zebrafish are indicated by green arrows. g Western blot of Ctrl and Tau zebrafish samples extracted by mechanical dissociation in tris-buffered saline without reducing agents or heating and probed with an antibody to total human Tau. The expected electrophoretic mobilities of monomeric human 0N/4R-Tau and additional high molecular weight forms corresponding to oligomers are indicated. Source data are provided as a Source Data file.
|
