Biochemical analysis of tau species in control and compound mutant young mice. The three columns display protein samples derived from litters of increasing ages (45, 60 and 70 days old). The human APP695 transgene (“HuAPP”) and Trp73 genotypes were confirmed by PCR-based methodologies (top two rows) as described in material and methods. Western blot analysis is shown for transgene-encoded human APP (row 3: 6E10 antibody), with total tau (row 4) and actin (row 5) immunoblots serving as a loading control in soluble fractions. Tau antibodies, AT8, AT180, CP13 and PHF-1 (rows 6-9, respectively), were used to evaluate pathological forms of tau in each sample in insoluble fractions. A tau transgenic sample (left-hand lane) was included in the immunoblot series for each time-point as a positive control (TgTauP301L mouse, age 540 days). “+/-“ in the case of Trp73 and TgAPP indicates that these mice were heterozygous for null allele and hemizygous the TgCRND8 APP transgene array, respectively. Loading controls (“Loading”; row 10) consist of images of Ponceau red-stained membranes taken immediately after blotting.