Western blots of TS-soluble and Sarkosyl-insoluble fractions of R406W brains and protein chemical analysis of PHF-tau purified from R406W brains. A: Specificities of AR406 and AW406 confirmed by Western blotting of recombinant wild-type (lanes 1–4) and R406W tau (lanes 5–8) as authentic antigens. Lanes 1 and 5, 0.8 ng; lanes 2 and 6, 4 ng; lanes 3 and 7, 20 ng; lanes 4 and 8, 100 ng; and τ (rightmost lane), six isoforms of recombinant tau. B: Sarkosyl-insoluble fractions prepared from frontal cortices of an AD patient (lane 1), R406W Patient 1 (lane 2), and Patient 2 (lane 3), temporal cortex of Patient 2 (lane 4), hippocampus of Patient 2 (lane 5) and cerebellum of Patient 2 (lane 6) were subjected to semiquantitative Western blotting with AR406 and AW406. PHF-tau (arrowheads) and a smear were labeled in specimens from various cortices of an AD and R406W brains, but not in those from cerebellum. In our hands, the 64-kd band of PHF-tau are usually resolved into closely spaced two bands. All samples corresponded to preparations from 0.5 mg wet weight tissue, except for a sample from Patient 1, which was from 2.5 mg wet weight tissue. C: TS-soluble fractions were prepared from frontal cortices (lanes 1–3) and cerebella (lanes 4–6) of an AD patient (lanes 1 and 4), R406W Patient 1 (lanes 2 and 5), and Patient 2 (lanes 3 and 6). Before or after dephosphorylation (AP), proteins equivalent to that contained in 1.0 mg tissue were subjected to semiquantitative Western blotting with AR406 and AW406. Note that wild-type tau but not mutant tau in the frontal cortex and cerebellum of R406W brains exhibited a slight but obvious mobility shift after dephosphorylation. Upper two bands in AP+ lanes probably come from indiscernible phosphorylated counterparts with slower mobility. D: The PHF-tau purified from R406W brains (a) and authentic recombinant wild-type (b) and R406W tau (c) were digested with API, and the produced peptides were separated on a Select B column as described in Materials and Methods. Note that the elution positions of peaks 1 and 2 (a) exactly correspond to those of peaks 3 (b) and 4 (c), generated from wild-type and mutant tau, respectively. E: The peptides in peaks 1–4 in D were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The amino acid sequence of the carboxy half of human tau is shown. The mutation is indicated by arrowhead, and the carboxy-terminal peptide (residues 396–438) is boxed.

Double-labeled NFTs in R406W brains. Tissue sections from the frontal cortex and hippocampal region of R406W brain from the Dutch (A-H) and American families (I and J) 16 were immunostained with Alexa 488-conjugated AR406 (green for wild-type tau) and Alexa 568-conjugated AW406 (red for mutant tau), as described in Materials and Methods. NFTs were labeled with both antibodies to a similar extent, indicating that wild-type and mutant tau coexist in NFTs in R406W brains. C–J: Confocal images of NFTs in the frontal cortex (C–E) and dentate gyrus (F–H) of Patient 2 from the Dutch family, and in the subiculum (I and J) of a patient from the American family. 16 E and H represent merged views of C and D and of F and G, respectively. Scale bars, 100 μm (A, B, I, and J) and 10 μm (C–H).

Hyperphosphorylation of R406W tau in PHF-tau. Purified PHF-tau was rechromatographed on an Aquapore BU300 column, with a shallow gradient of acetonitrile (A). Western blotting with AR406 and AW406 showed substantial separation between wild-type and R406W tau (inset, A). B: Each of fractions 1–3 was subjected to Western blotting with AT8 (a), AT100 (b), M4 (c), C5 (d), PHF1 (e), and AP422 (f) to evaluate the extent of phosphorylation of wild-type (fraction 1) and R406W tau (fraction 3). Western blotting using tau 1 after dephosphorylation (g) represents the amounts of all tau isoforms in each fraction.