Figure 2: Lack of PBS-soluble phosphorylated HMW tau species is associated with low tau uptake in primary neurons.
(a, top) Uptake of human tau from brain extracts from rTg4510 and rTg21221 mice by primary neurons (PBS-3,000g, 500 ng ml−1 human tau). Neurons were immunostained with human tau-specific antibody (green) and total (human and mouse) tau antibody (red). (a, bottom) Tau uptake assay in HEK-tau-biosensor cells. Brain extracts (10 μg protein) were applied to the cells (lipofectamie (−)). (n=4) Unpaired t-test. Scale bar, 50 μm. (b) Human tau levels in brain extracts (ELISA). (c) Immunoblot analysis of PBS-soluble extracts with total tau antibody (DA9). Up-shifted bands in rTg4510 brain suggest phosphorylation of tau (arrow). (d) Brain extracts were immunoblotted with phospho-tau specific antibodies recognizing different epitopes. Representative immunoblot and quantification of phospho-tau levels at each epitope. (n=3–4) Unpaired t-test. (e,f) SEC analysis of PBS-soluble tau. (e) Representative graph of human tau levels (ELISA) in SEC-separated samples (f) Mean human tau levels of HMW (Frc. 2–4) and LMW (Frc. 13–16) SEC fractions. (n=3–6) Unpaired t-test. (g) Immunoblot analysis (SDS-PAGE) of SEC-separated fractions from brain extracts (total tau, DAKO). Quantification of band density is also shown (right graphs) (n=4). Unpaired t-test. (h) Dot blot analysis of PBS-soluble brain extracts with tau oligomer-specific antibody (T22), human tau-specific antibody (Tau13), and total tau antibody. Quantification of dot blot signals is also shown (right) (n=4). Unpaired t-test. Eleven to thirteen-month-old animals were used. *P<0.05, **P<0.01.
