Fig.Figure 3(A and B) Expressing Tau inhibited β-cat ubiquitination in HEK293 cells, measured by immunoprecipitation using antibody against β-cat and Western blotting using anti- ubiquitination (anti-Ub) and anti-β-cat, respectively (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec (B) Data were analyzed by Student's t test. (C and D) Overexpressing Tau inhibited β-cat ubiquitination in hippocampi of Tau transgenic mice, measured by immunoprecipitation using anti-Ub and Western blotting using anti-β-cat, respectively (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau- (D) Data were analyzed by Student's t test. (E and F) Expressing Tau-k18(-) attenuated Tau-induced β-cat elevation. HEK293 cells were transfected with Vec, or Tau-40, or Tau-k18(-) for 24 h, and then were treated with Chx for 12 h or 24 h, followed by Western blotting. β-cat protein level was normalized to β-actin (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau (F) Data were analyzed by Two-way repeated-measures ANOVA (G and H) K49 acetylation of β-cat inhibited its ubiquitination. HEK293 cells were co-transfected with HA-ubiquitin and GFP-β-cat or pseudo-acetylated K49-β-catenin (K49Q), and then β-cat was immunoprecipitated by anti-GFP and blotted by anti-Ub and anti-β-cat (n=3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT Data were analyzed by Student's t test. (I and J) K49 acetylation of β-cat inhibited its degradation. HEK293 cells were transfected with β-cat WT or K49Q for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT Data were analyzed by Two-way repeated-measures ANOVA β-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). β-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau+β-cat WT (L) Data were analyzed by Two-way repeated-measures ANOVA (M and N) K49 acetylation of β-cat inhibited its phosphorylation at N-terminal domain. HEK293 cells were transfected with GFP-vec, or eGFP-β-cat (WT) or K49Q for 48 h, then treated with wortmannin (WO, 1 μM) plus GF109203X (GFX, 1 μM) to activate GSK-3β for 1 h followed by Western blotting (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT plus WO/GFX (N). data in (N) was analyzed by one-way ANOVA.