Fig. 3: TauC3 Ser416 phosphorylation regulates CHIP-dependent tau homeostasis.

A In vitro ubiquitination of tau proteoforms by CHIP. Samples were collected at the denoted timepoints, quenched in SDS-PAGE loading buffer, and analyzed by western blot. B Quantification of unmodified tau remaining following in vitro ubiquitination of varying tau proteoforms by CHIP. Unmodified tau remaining was analyzed by densitometry, normalized to 0-min time point, and curves were fit using one-phase exponential decay (n = 3 technical replicates). C Cartoon schematic depicting promoter architecture and varying C-terminal sequences for HEK293 FlpIn T-Rex cells expressing doxycycline inducible GFP-tau proteoforms. D Representative fluorescence micrographs for GFP-tau cells. Images show tau species associated with microtubules (green, false color), while nuclei are stained with Hoechst 33342 (blue, false color). Scale bar = 30 µm. Images are representative of three separate acquisitions. E Co-immunoprecipitation assay following IP of varying tau species from cells. Whole cell lysate (input) was used for loading controls, and co-immunoprecipitated CHIP was analyzed by western blot. Experiment was repeated in duplicate. F Representative western blot showing differing abundance of various tau proteoforms in HEK293 FlpIn T-Rex cells. Experiment was repeated in duplicate. G Quantification of tau protein abundance taken from three independent biological experiments. Tau:tubulin ratio was determined by densitometry and normalized to full-length tau. Error bars represent SD. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc analysis. H Quantification of MAPT mRNA from three independent biological experiments. MAPT mRNA was normalized to GAPDH and shown relative to full-length tau. Error bars represent SD. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc analysis.