![Figure 3. 14 amino acids in the tail of KIF1C mediate its interaction with Hook3. (A) Schematic of constructs used to map the region of KIF1C that is responsible for binding to Hook3. (B) KIF1C-SNAP-3xFLAG constructs were transiently expressed in 293T cells and immunoprecipitated with FLAG affinity resin (FLAG-IP). Immunoblots were performed with anti-Hook3 and anti-FLAG antibodies. 3xFLAG-sfGFP provided a control. Protein molecular weight markers are shown in kilodaltons on the anti-FLAG immunoblot. (C) Representative kymographs from single-molecule motility assays with purified KIF1CΔ794-807-TMR in the presence of Hook3-488. Microtubule polarity is marked with minus (–) and plus (+). (D) Schematic of constructs used to map the region of Hook3 that is responsible for binding to KIF1C. Hook3NT (aa 1–552), Hook3CT (aa 553–718), Hook3Hook2 (a Hook3 [aa 1–552] and Hook2 [aa 548–719] chimera). (E) HaloTag-Hook3-3xFLAG constructs were transiently expressed in 293T cells and immunoprecipitated with FLAG affinity resin (FLAG-IP). Immunoblots were performed with anti-KIF1C and anti-FLAG antibodies. 3xFLAG-sfGFP provided a control. Protein molecular weight markers are shown in kilodaltons on the anti-FLAG immunoblot. (F) Representative kymographs from single-molecule motility assays of KIF1C-TMR in the presence of Hook3Hook2-488. Microtubule polarity is marked with minus (–) and plus (+).](https://cdn.rupress.org/rup/content_public/journal/jcb/218/9/10.1083_jcb.201812170/8/jcb_201812170_fig3.jpeg?Expires=1773828820&Signature=DvUo0RKA9bupRkimMMQzzjPQauw9W20c2aW3gCShILZfl~VWbECL4RBj~JTMN63ghfbLnQDCRYh7L3ziSmx0ixrD7AperP63KWleFnitkXgyuLenMpow-15620iTP9PVBDBnFqAQJ3BqnC-mj91CHXaxgwg4PRqewYFuEQHXUJndqDWS4IKYZkpDfkx4eV5rAIwQMh-2S0-X1dr55tYR~kdriztgn36pCbV~3fVWtwh9dHyoKc0BsRKH4~W6sEvIXxG~Nnwc6357RzEBlBKaYKxOBsZnozld~w8E1x79wEa5CeUJAP0ifiCM05esXm54xIHqleiWRugsVYAG9E-x9A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
14 amino acids in the tail of KIF1C mediate its interaction with Hook3. (A) Schematic of constructs used to map the region of KIF1C that is responsible for binding to Hook3. (B) KIF1C-SNAP-3xFLAG constructs were transiently expressed in 293T cells and immunoprecipitated with FLAG affinity resin (FLAG-IP). Immunoblots were performed with anti-Hook3 and anti-FLAG antibodies. 3xFLAG-sfGFP provided a control. Protein molecular weight markers are shown in kilodaltons on the anti-FLAG immunoblot. (C) Representative kymographs from single-molecule motility assays with purified KIF1CΔ794-807-TMR in the presence of Hook3-488. Microtubule polarity is marked with minus (–) and plus (+). (D) Schematic of constructs used to map the region of Hook3 that is responsible for binding to KIF1C. Hook3NT (aa 1–552), Hook3CT (aa 553–718), Hook3Hook2 (a Hook3 [aa 1–552] and Hook2 [aa 548–719] chimera). (E) HaloTag-Hook3-3xFLAG constructs were transiently expressed in 293T cells and immunoprecipitated with FLAG affinity resin (FLAG-IP). Immunoblots were performed with anti-KIF1C and anti-FLAG antibodies. 3xFLAG-sfGFP provided a control. Protein molecular weight markers are shown in kilodaltons on the anti-FLAG immunoblot. (F) Representative kymographs from single-molecule motility assays of KIF1C-TMR in the presence of Hook3Hook2-488. Microtubule polarity is marked with minus (–) and plus (+).
