Fig. 3. Diurnal oscillations in tau protein solubility in relation to the expression of Hsp70 in the hippocampus of P301S transgenic mice. (A) The western blot using the serially extracted protein extracts and its densitometry analysis shows the circadian alterations of tau protein levels, both phosphorylated- and total tau, showing a specified pattern according to the status of tau solubility. ZT14 is specifically identified as the highest ZT point for the RIPA-soluble tau levels while the lowest time-point for the RIPA-insoluble tau protein at the same time. Data are mean±SEM (n=3). #p<0.1, *p<0.05 and, **p<0.01 on Kruskal-Wallis test adjusted for multiple comparisons using Dunn’s post-hoc correction. (B) The ZT-associated alterations in Hsp70 protein expression in P301S mice are shown on western blot and densitometry analysis in comparison to wild-type mice. Data are mean±SEM (n=3). *p<0.05 on Kruskal-Wallis test followed by Dunn’s post-hoc correction. (C) The circadian variability in Hsp70 protein levels is a similar pattern to that of the RIPA-soluble tau, but an opposite pattern to the RIPA-insoluble fraction suggesting the possible role of Hsp70 in tau disaggregation (left). The highest difference is noted at ZT14. Data are mean±SEM (n =3). *p<0.05 and **p<0.01 on Kruskal-Wallis test followed by Dunn’s post-hoc correction. Spearman’s correlation analysis (right) shows the negative correlation between Hsp70 and tau (rs=-0.7343, p<0.01). (D) Immunohistochemistry of cortical brain sections indicates co-localization of Hsp70 with tau proteins. Scale bars are 100 μm or 10 μm as indicated. CTX, cortex; FA, formic acid.
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