Fig. 2: Phosphorylation of tauC3 Ser416 is sufficient to inhibit interaction with CHIP.
From: Phosphorylation of tau at a single residue inhibits binding to the E3 ubiquitin ligase, CHIP
A Cartoon depicting constructs used for mapping of inhibitory phosphorylation sites by NanoBiT live cell assays. B NanoBiT live cell assay for CHIP interactions with truncated tau constructs following treatment with okadaic acid (30 nM, 18 h) or DMSO control. Data are shown as the difference between okadaic acid treated samples and vehicle control. Error bars represent SD. Statistical significance was determined by two-sided, one-way ANOVA with Tukey’s post hoc analysis (n = 3 biological replicates). C Apparent change in melting temperatures (Tmapp) for CHIP incubated with DMSO control or Hsp70 C-terminal peptides, as determined by DSF. Error bars represent SD. (n = 4 technical replicates). D DSF experiments performed and analyzed as in the previous panel, but with tauC3-derived peptides. (n = 4 technical replicates). E Competition FP experiment showing displacement of fluorescent tracer from the CHIP TPR domain by various 10-mer Hsp70 or tauC3-derived peptides. Experiments were performed in technical quadruplicate. F Inhibition constants derived from (E). Error bars represent SD. G Tau proteoforms binding to immobilized CHIP, as measured by ELISA. Assay was performed in technical triplicate and normalized to maximum absorbance at 450 nm. H Dissociation constants derived from (G). Error bars represent SD.
