Figure 2.

Immunoblot analysis of RAB soluble human MAPT and mouse Mapt isoforms in brains from wildtype and transgenic mice. Whole brain homogenates were prepared as described in the Materials and Methods and subjected to electrophoresis and immunoblot analysis using the following antibodies: RD4 (1-1000 dilution, top panels, A and B); RD3 (1-3000 dilution, middle panels, A and B); Rb17025 (diluted 1-6000, bottom panel, A and B) which recognizes both mouse and human tau (all six isoforms); T14 (1-1000) (C, all panels), an antibody specific for human tau (all six isoforms) that does not recognize mouse tau. Protein loads/lane for A and B are: 1.2 ug, top panel; 0.1 ug, middle panel; 0.2 ug, bottom panel. Lanes labeled with “-” are untreated soluble tau and lanes labeled “+” were treated with λ-phosphatase. A. Wildtype mice produce predominantly 3R tau at P6, both 3R and 4R tau at P24 and predominantly 4R tau at P90. Note that for all isoforms, the mouse protein migrates faster than the human equivalent protein. However, spacing is not altered. B. The human gene in a Mapt^-/- mouse produces predominantly 3R tau and no 4R tau at P6. By P24, other tau isoforms were observed including some that are 4R, although the predominant isoform is still 3R even in the adult mice (P90). C. The human gene in the adult mouse brain produces all 6 tau isoforms and FTDP-17 mutations that alter isoform ratios in man also affect splicing of E10 in mouse. RAB soluble whole brain homogenates were prepared from mice transgenic for either the normal MAPT gene (hT-PAC-N) or MAPT with the FTDP-17 mutations ^V337^M, ^R5^L, ^S305^S, E10+14, ^P301^L, or ^N279^K on a mouse Mapt⁺ background. Samples in the top panel were untreated and samples in the bottom panel were treated with λ-phosphatase. Since different transgenic lines produce different amounts of human tau, loading was adjusted so that approximately the same amount of human tau was present in each lane. The protein loaded for phosphatase treated samples was as follows: 0.25 ug, hT-PAC-N; 0.17 ug, hT-PAC-337^M; 0.43 ug, hT-PAC-5L; 0.9 ug, hT-PAC-305^S; 0.18 ug, hT-PAC-E10+14; 0.2 ug, hT-PAC-301^L; and 0.2 ug, hT-PAC-279^K. The protein loaded for untreated samples was 0.5 ug, except for hT-PAC-337^M, which was 0.1 ug. The standards are recombinant human tau.