Research Paper Volume 7, Issue 10 pp 839—853

Figure 4. MAP1S interacts with HDAC4 via the HDAC4-binding domain (HBD) in the overlapping region between the HC and SC of MAP1S.

(A) A diagram showing the sequence domains of MAP1S proteins. FL, full length; HC, heavy chain; SC, short chain; LC, light chain; 4G1, region recognized by MAP1S monoclonal antibody 4G1; HBD, HDAC4-binding domain within the R653-Q855 fragment. All sequence numbers were deduced based only on our experimental results. (B) Endogenous HDAC4 interacts with MAP1S. HeLa cell lysates were precipitated with a MAP1S-specific antibody 4G1 or IgG control antibody. (C, D) Endogenous HDAC4 interacts with MAP1S. Lysates from brain tissues of wild-type (+/+) and MAP1S−/− mice (−/−) (C) or N2a cells (D) were precipitated with a specific antibody against HDAC4 or MAP1S, or IgG control antibody. (E, F) Interaction of HDAC4 and MAP1S in cells overexpressing both HDAC4 and MAP1S FL. In (E) lysates from 293T cells overexpressing HA-MAP1S were precipitated with HA or MAP1S-specific antibody or IgG control. In (F) lysates from normal 293T cells and 293T cells overexpressing both HDAC4 and HA-MAP1S were precipitated with MAP1S-specific antibody or IgG control. (G) HDAC4 interacts with MAP1S isoforms SC, HC and FL. Lysates from 293T cells overexpressing HDAC4 and HA-fused MAP1S isoforms were precipitated with MAP1S-specific antibody or IgG control. (H) HDAC4 interacts with MAP1S SC but not LC. Lysates from 293T cells overexpressing HDAC4 and HA-fused MAP1S SC and LC were precipitated with a HA-specific antibody or IgG control. (I, J) HDAC4 in cell lysates (I) is pulled down by GSH beads bound with purified GST-fused MAP1S SC, but not LC (J). HDAC4 was revealed on blots with specific antibody and GST fusion proteins were visualized with Coomassie Blue staining. (K) HDAC4 interacts with an overlapping domain between MAP1S HC and SC. Lysates from 293T cells overexpressing HDAC4 and HA-fused MAP1S sequence R653-Q855 (HA-HBD) were precipitated with a HA-specific antibody or IgG control and blotted with HA or MAP1S-specific antibody. (L) The Interaction of MAP1S with HDAC4 is abolished when the HBD is deleted. Lysates from 293T cells overexpressing HDAC4 and HA-fused full length MAP1S (HA-FL) or mutant with sequence R653-Q855 deleted (HA-HBDΔ) were precipitated with a HA or HDAC4-specific antibody or IgG control and blotted with HA and HDAC4 antibodies.