Fig 2: LC-MS/MS reveals Tau phosphorylation. Primary sequence of human Tau441 with highlighted sequence coverage (81%; letters in black) and phosphorylation sites (letters in red) revealed by LC-MS/MS. The microtubule binding domain of Tau is underlined.

Fig 3: MAP kinase p38a is a multi-site tau kinase in vivo.(A) Tau SP/TP phosphorylation interdependence is kinase specific. Directionality of interdependence relations between initial phosphorylation (a) and modulated phosphorylation (b). Connecting line thickness indicates significance and modulation strength. Kinases with extensive high-significance interdependence (GSK3ß and p38a) and kinases with sparse relations (Cdk5 and JNK2) are shown. n = 4. N, N terminus; C, C terminus of tau (441 amino acids) primary sequence. (B) Correlation matrix of tau phosphorylation interdependence relations associated with 12 SP/TP kinases (n = 4), including hierarchical clustering by pair-wise Pearson’s correlations. Note that tau kinase similarity differs from evolutionary relation of kinase families for GSK3 and p38 kinases. (C to E) Phosphorylation of endogenous tau is reduced at multiple epitopes in hippocampus of mice with neuron-restricted p38a deficiency (p38a?neu) as compared with control p38aloxP/loxP mice. (D) Representative immunoblots probed with antibodies for pS199 tau, pS202 tau, pS396 tau, pS404 tau, pS422 tau, tau, and p38a are shown. n = 6. Residual p38a in p38a?neu is due to expression in glial cells (36). (E) Immunoblots in (D) were quantified relative to Gapdh and tau. Data are expressed as means ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05. Note that most phosphorylation sites tested show markedly lower levels in p38a?neu as compared with control p38aloxP/loxP mice.

Fig 4: Tau does not directly interact with P53.Cell lysates of SH-SY5Y treated with 10 µM of MG132 to stabilize P53 expression, without (ctrl) or with (eto) a 30 min pre-treatment with 60 µM etoposide, were subjected to immune precipitation of endogenous P53 with a rabbit antibody (P53) or with a rabbit GFP antibody as negative IP control (GFP). Western blot analysis for co-precipitation of MDM2 or Tau with the respective mouse antibodies as indicated. The blots on the top show the analysis of the starting material (cell lysates), those on the bottom the immunoprecipitation (IP). The P53 blots are entirely shown, whereas for MDM2 and Tau, the blots were cut between the 55 kDa and the 95 kDa protein size markers and analyzed separately.

Fig 5: Reduced P53 in Tau-KO cells is not caused by DDR activation.For all panels, parental (wt) or 232P (Tau-KO) cells were treated 30 min with the indicated etoposide concentrations and recovery times. a Mean intensity ± SD of ?H2A-X staining normalized for DAPI staining by in-cell western is shown as fold of wt cells at basal conditions, n = 5 of biological replicates in a 96-well plate. Non-parametric independent Mann–Whitney U test between lines (in bold), or for each dose between lines (in italics). b Olive moment in the Comet assay is shown as mean ± sem, n = 84–146 cells/condition. c, d Mean intensity ± sem of single-cell nuclear ?H2A-X, pATM or pChk2 staining (DAPI mask, ImageJ) is shown as fold of wt cells at basal conditions, n > 100 cells/condition distributed over five images. Statistical analysis by independent measures ordinary two-way ANOVA, source of variation for cell lines (in bold), multiple Bonferroni pairwise comparisons of each condition between lines (in italics) and of time points for each line (b, d, in vertical).