Fig. 8

ADNP indirectly interacts with the histone deacetylase enzyme SIRT1 through the microtubule end-binding proteins EB1/EB3. (A) Adnp and Sirt1 immunostaining (red Cy3 fluorescence) in cryosections of the murine cerebellum was assessed by confocal scanning microscopy. Adnp was mainly observed in the nucleus and Sirt1 was mostly located in the cytoplasm. (B) Co-IP assay of Adnp and Sirt1 in the murine cerebellum. IP-competent EB1 and EB3 antibodies were crosslinked to agarose beads and sequentially eluted in fractions (input, In; flow-through, Ft; three consecutive washes, w1-w3; and elution, E). All fractions were analyzed by western blotting for Adnp, Sirt1, EB1, and EB3. IgG non-reactive beads were used as a negative control. GAPDH has been used as loading control for all western blots, and critical assessment of the accuracy of the Co-IP method. (C) ELM analysis identified shared motif sequences of Adnp and Sirt, including 14–3–3 motifs (green), SxIP motif (blue), SH3 domains (orange), and the SSIP motif (violet). (D) In silico 3D-molecular docking of Adnp (SxIP motif) to EB1/3, left (purple) and right (pink) respectively to Sirt1 in the panel below (SSIP motif) to EB1/3, left (purple) and right (pink)