JIP1 binds directly to both stalk and tail domains of KHC independently of KLC. (A) JIP1 coimmunoprecipitates with KHC in mouse brain homogenate. A monoclonal JIP1 antibody immunoprecipitates the expected 110-kD band as well as a 90-kD band that likely represents a splice isoform. Both bands are also recognized by multiple JIP1 monoclonal antibodies (R&D Systems and BD). (B) Schematics of KHC (KIF5C) and JIP1 constructs and summary of mapping results. JIP1 contains JBD (JNK-binding domain), SH3 (src homology), and PTB domains. (C) JIP1 binds to both stalk and tail domains of KHC. Lysates from COS7 cells cotransfected with myc-JIP1 and GFP-KHC fragments were immunoprecipitated with an anti-myc antibody. (D) JIP1 binds directly to KHC stalk. Purified His-JIP1 incubated with glutathione beads bound to either GST or GST-KHC-stalk (aa 560–682) selectively bound to GST-KHC-stalk but not GST. (E) JIP1 binds directly to KHC tail. Purified His-KHC-tail (KIF5B aa 823–944) with or without His-JIP1 was coincubated with anti-JIP1 antibody, which specifically coimmunoprecipitated His-KHC-tail. (F) KHC stalk binds to the C terminus of JIP1 independently of KLC. Lysates from COS7 cells cotransfected with GFP-KHC-stalk and myc-JIP1 fragments were immunoprecipitated with an anti-myc antibody. KHC stalk coimmunoprecipitated with myc-JIP1[554–711] (myc-JIP1-SBD). Asterisk shows antibody light chain bands. (G) KHC tail binds to JIP1 independently of KLC. Lysates from COS7 cells transfected with GFP-KHC-tail and myc-JIP1 fragments were immunoprecipitated with an anti-myc antibody. KHC tail coimmunoprecipitated with myc-JIP1[285–440] (myc-JIP1-TBD). Asterisk shows antibody heavy chain bands.