Fig. 2

Ttbk1 ASOs suppresses pS422 tau level in PS19 mice. A Protein extraction scheme. B Western blot results against pS422 and total tau (tau5) antibodies with soluble fraction S1 isolated from the hippocampal tissue 8 weeks after the injection of ASO-Ttbk1 or -control. C: WT mouse sample H: WT mouse samples under hypothermia to induce tau phosphorylation as a positive control, M: Marker, 1–8: Samples from PS19 mice injected with ASO-control, ASO-Ttbk1#1, or ASO-Ttbk1#2. C Protein expression levels of pS422 normalized by total tau. All membranes were always loaded with a constant amount of the same positive control (PC) at the same time, and all bands of pS422 and tau5 were quantified as a ratio to PC. One-way ANOVA and Dunnett’s multiple comparison. * denotes < 0.05. D Association analysis of pS422 level normalized by total tau and TTBK1 protein level in S1 fraction of the hippocampal tissues measured by ELISA. The GraphPad was used to confirm the Gaussian distribution, and then simple liner regression analysis was performed. E Representative immunofluorescence image of phosphorylated tau (pS422) in the hippocampal region in PS19 mice injected with ASO-control, ASO-Ttbk1#1, or ASO-Ttbk1#2 at 8 weeks post injection. pS422 (red) and DAPI (blue) immunostaining in the CA1 and mossy fiber. Scale bar = 100 μm. F Mean intensity measurement of pS422 in the mossy fiber region. **p < 0.01 compared to ASO-control treatment group as determined by One-way ANOVA and Dunnett’s multiple comparison. G Quantification of pS422 positive neuronal volume normalized by total CA1 volume (ASO-control n = 7; ASO-Ttbk1 #1 n = 6; ASO- Ttbk1 #2 n = 8 mice)