FIGURE 2:

Mutation of phosphorylation sites did not reduce Tau dimerization under conditions of long-term mitochondrial stress. (A) PP2A phosphatase activity measured with the malachite green phosphatase assay in HEK293T wild-type cells and HEK293T NDUFA11 knockout cells. The data are expressed as mean ± SEM. n = 3. (B) Phosphorylation of Tau protein determined by immunoblotting with phospho-Tau (Ser-202, Thr-205) antibody in HEK293T wild-type cells and HEK293T NDUFA11 knockout cells. The data are expressed as mean ± SEM. n = 3. (C) Schematic illustration of BiFC assay. Cells were transfected for 72 h with a pair of BiFC vectors that encoded wild-type (WT) Tau (Tau-VN and Tau-VC) or a pair of BiFC vectors that encoded mutated Tau (Tau Ser/Thr phosphomutants; Tau-VN-AP and Tau-VC-AP). Tau was mutated to alanine in all 14 S/P or T/P amino acid residues (T111, T153, T175, T181, S199, S202, T205, T212, T217, T231, S235, S396, S404, and S422; numbering based on the longest 441-amino-acid brain isoform of hTau). BiFC plasmids encoded Tau protein that was fused with the N-terminal part of Venus protein (Tau-VN, Tau-VN-AP) or Tau protein that was fused with the C-terminal part of Venus protein (Tau-VC, Tau-VC-AP). The dimerization and oligomerization of Tau protein enabled the reconstitution of functional Venus protein, resulting in an increase in fluorescence. (D) Phosphorylation of Tau protein determined by immunoblotting with phospho-Tau (Ser-202, Thr-205) antibody in HEK293T wild-type cells and HEK293T NDUFA11 knockout cells. Cells were transfected with BiFC vectors that encoded wild-type (WT) Tau (Tau-VN and Tau-VC) or BiFC vectors that encoded Tau, in which all 14 S/P or T/P amino acid residues (T111, T153, T175, T181, S199, S202, T205, T212, T217, T231, S235, S396, S404, and S422; numbering based on the longest 441-amino-acid brain isoform of hTau) were mutated to alanine (Tau Ser/Thr phosphomutants; Tau-VN-AP and Tau-VC-AP). n = 3. (E) Fold change in Venus fluorescence normalized to the level of Tau expression in HEK293T wild-type (WT) cells and HEK293T NDUFA11 knockout (KO) cells. Cells were transfected with BiFC vectors that encoded wild-type Tau (Tau-VN and Tau-VC) or BiFC vectors that encoded Tau, in which all 14 S/P or T/P amino acid residues (T111, T153, T175, T181, S199, S202, T205, T212, T217, T231, S235, S396, S404, and S422; numbering based on the longest 441-amino-acid brain isoform of hTau) were mutated to alanine (Tau-VN-AP and Tau-VC-AP). The data are expressed as mean ± SEM. n = 4. *p < 0.05, **p < 0.01; ns, not significant (p > 0.05) (Student’s t test or one-way ANOVA followed by Tukey’s multiple comparisons test in E).