Figure 6
Validation of pS404, pT231 and 3R tau. (A) Immunoblotting of sarkosyl-insoluble tau with rabbit primary antibody against phospho-Ser404 (1:200; OAAF07796, Aviva Systems Biology). The entire membrane is shown. Hyperphosphorylation at the S404 residue was exclusively observed in 24-month-old APPswe/PS1ΔE9 mice (TG 24 M, lane 8). Lanes are labelled as follows: Marker: 1, 14; AD: 3; non-AD: 5; WT 24 months: 6; TG 24 months: 8; WT 3 months: 10; TG 3 months: 12; Empty: 2, 4, 7, 9, 11, 13. (B) ELISA of pT231 tau. Soluble pT231 tau was present in the neocortex of both WT and TG mice. Phosphorylation at T231 was only observed in the sarkosyl-insoluble fraction from 24-month-old APPswe/PS1ΔE9 mice. Results are expressed as arbitrary units (U), normalized to total protein concentration. (C) Immunohistochemistry of pT231 tau in coronal, 50 µm-thick brain sections from 3- and 18-month-old WT and TG mice. Black arrows point to CA1 pyramidal neurons, which were more strongly immunolabelled in 18 vs. 3-month-old animals, irrespective of genotype. Scale bar: 200 µm. The inserts show higher magnifications of layer V neurons in the temporal cortex, with black arrowheads pointing to tangle-like structures in aged APPswe/PS1ΔE9 mice. Note reduced dendritic staining in 18 vs. 3-month-old animals. Scale bar: 10 µm. White arrows point to puncta of pT231 immunoreactivity within a plaque-like structure, observed exclusively in the neocortex of TG mice. Scale bar: 20 µm. Abbreviations: CA1: cornu ammomis field 1; ec: external capsule; o- striatum oriens; p: striatum pyramidalis; r: striatum radiatum. (D) ELISA of 3R tau. Low levels of soluble 3R tau were present in the neocortex of both WT and TG animals. Sarkosyl-insoluble 3R tau was only observed in 24-month-old APPswe/PS1ΔE9 mice. Results are expressed as arbitrary units (U), normalized to total protein concentration.
