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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: J Alzheimers Dis. 2013 Jan 1;33(1):249–263. doi: 10.3233/JAD-2012-121093

Fig. 3. Tau phosphorylation in TBS-extractable fractions from rTg4510 mouse brains.

Fig. 3

A. Western blots of TBS-extractable fractions from rTg4510 (tg) and non-tg (nt) mice aged 4 months (4 M) and 8 months (8 M) using 12 tau-specific antibodies. The same volume (containing 0.01 mg wet-weight of brain) of each sample was loaded into the gels. Arrowheads indicated 64 kDa tau. Bold lines indicated 50–60 kDa tau. B. Western blots of TBS-soluble tau derived from the brains of 2, 4, 6, and 8 month-old rTg4510 mice. Blots were probed with E1, 12E8, and PHF1 antibodies. Arrowheads indicated 64 kDa tau. C. Temporal changes in tau phosphorylation with aging. Relative phosphorylation levels detected by 12E8 and PHF1 antibodies were normalized by the levels of E1-positive tau. Values are means ± SEM with respect to levels measured from samples of 2 month-old mice; these levels were scaled to a value of one. D. Two-dimensional SDS-PAGE characterization of 64 kDa tau. Fifteen micrograms of S1 fraction from the brain of a 6 month-old rTg4510 mouse was separated via isoelectric focusing at a pH range of 4–7 (D) or 3–10 (E) for the first dimension, and then via 10% SDS-PAGE for the second dimension. The resulting blots were probed with anti-tau antibody WKS44 and anti-phosphorylated tau specific antibody PHF1. The pI of 64 kDa tau species was between 5.0 and 6.0. Arrows indicated molecular size of 64 kDa in the second dimension. Isoelectric point determination was validated using loading controls (e.g., β-tubulin, β-actin) and ponceau staining (E).