Figure 8.

CHIP-deficient mice overexpressing a mutant form of human tau convert all human tau into a higher molecular weight species but still fail to accumulate insoluble tau species. Whole brain was extracted from 21-d-old CHIP^+/+, CHIP^−/−, CHT, and JNPL3 mice (n = 3 per group, 2 shown per group), and homogenates were fractionated using Sarkosyl solubilization for SDS-PAGE and Western blot analyses. A, Membranes were probed with the following tau antibodies: pS396/S404-tau (PHF1), total tau (Tau5), dephosphorylated at S199/S202 (Tau 1), and human-specific total tau (E1). CHT mice had a similar tau profile to CHIP^−/− mice in that phospho-tau and total tau levels were elevated and dephosphorylated tau was reduced. Note the absence of phospho-tau species in age-matched JNPL3 mice compared with CHT mice. CHT mice probed with the human-specific total tau antibody detected similar expression levels compared with JNPL3 mice; however, the molecular weight of tau was higher in the CHT mice, which collapsed with alkaline phosphatase (PPase) treatment. GAPDH levels were unchanged. B, Coimmunoprecipitation (IP) of tau, followed by immuno-probing for polyubiquitinated species, demonstrated that the tau accumulating in the CHT mice is ubiquitin negative, whereas age-matched JNPL3 littermates did exhibit some polyubiquitination of tau. For blots, values shown on the right are in kilodaltons.