SNPH interacts with KIF5. (A) GST-SNPH (203–469 aa) pulls down the kinesin-1 heavy chain (KHC or KIF5) from mouse brain homogenates (BH). (B) An anti-SNPH antibody, but not preimmune serum (PIS), immunoprecipitates KHC from brain homogenates. KLC and the dynein intermediate chain IC74 are not detected in the same assays. (C) Coimmunoprecipitation showing a native SNPH–KIF5 complex in mouse brains. SHPN was pulled down by an anti-KHC antibody from wild-type (WT) mouse brain homogenates followed by detection with an anti-SNPH antibody. As a control, the same antibody failed to detect the SHPN–KIF5 complex from snph knockout (KO) mouse brains. (D) GST pull-down showing SNPH binding to the C-terminal tail (814–963 aa) of KIF5B. GST-SNPH (203–469 aa) or GST was incubated with His-tagged KIF5B truncated mutants. The bound protein complexes were blotted with an anti–H7-His antibody. Coomassie blue staining shows the relative purity of the recombinant proteins. (E) Schematic diagram of truncated SNPH mutants. MTB, MT binding domain; KBD, KHC binding domain; Mito, mitochondrial targeting sequence. (F) Coimmunoprecipitation showing KIF5 binding to the SNPH sequence (381–466). GFP-KIF5C and GFP-SNPH truncated mutants were coexpressed in HEK293 cells. The cell lysates were immunoprecipitated (IP) with an anti-KHC antibody and immunoblotted (IB) with an anti-GFP antibody. Note that the 86-residue segment (381–466 aa) of SNPH is sufficient to bind to KIF5C and is thus referred to as the KBD. (G) Deleting KBD abolishes SNPH binding to KIF5C. GFP-tagged KIF5C, SNPH, and its KBD deletion mutant, or KBD alone, were coexpressed in HEK293 cells. The cell lysates were immunoprecipitated with an anti-KHC antibody and immunoblotted with an anti-GFP antibody. Red boxes indicate SNPH, its KBD deletion mutant, or KBD alone. Asterisks show degraded bands.