Figure 4.

DNRX binds with the PDZ domains of Scribble via the C-terminal PDZ-binding motif. A, schematic representation of the protein structure of DNRX and Scribble. The polypeptide structure of the GST-fused DNRX C-terminal section with and without the C-terminal PDZ-binding motif and the PDZ domains of Scribble with HA and HIS tags. B, schematic graph of full-length HA-tagged Scribble of Drosophila and HIS-tagged α-Neurexin of mouse constructs. C, immunoprecipitated (IP) from whole Drosophila adult brain extracts with GFP nano-antibody fused beads in scribble::gfp and wild-type strains, respectively. Anti-GFP antibody precipitated the DNRX from the lysates, whereas the control IgG and wild-type sample did not. Suggesting DNRX physically interacts with Scribble in vivo. Input is 1%. D, Western blots of anti-His immunoprecipitates using protein lysate of HEK293 cells that can express the proteins of HA-Scribble and HIS-α-Neurexin, showing that HA-Scribble was coimmunoprecipitated with HIS-α-Neurexin in mouse. E, Coomassie Brilliant Blue for the purified DNRX C-terminal protein and deleted final 7-amino acid protein. F, Western blot validation for the purified 4 PDZ domain polypeptides of Scribble. G and H, GST pulldown with the DNRX C-terminal protein and deleted a very C-terminal 7-amino acid protein, respectively. Incubating with 4 PDZ domains of Scribble. Western blot results showed DNRX directly binds with all the PDZ domains of Scribble.