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. 2019 Jul 18;218(9):2982–3001. doi: 10.1083/jcb.201812170

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© 2019 Kendrick et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 1. — Endogenous Hook3 and KIF1C interact specifically. (A) Domain organization of KIF1C and Hook3. KIF1C contains an amino-terminal kinesin motor domain and regions of predicted coiled coil (CC), a forkhead-associated domain (FHA), and a proline-rich (P-rich) region in its carboxy-terminal tail. Hook3 is largely made up of regions of predicted CC and contains dynein/dynactin and KIF1C-binding regions (McKenney et al., 2014; Redwine et al., 2017). The Hook domain, which is also involved in dynein binding (Schroeder and Vale, 2016), is indicated. (B) 293T cells were transfected with control CRISPR/Cas9 (CTRL) or with CRISPR/Cas9-gRNA specific for KIF1C. KIF1C knockout (KIF1C^KO) was confirmed in two different clones by immunoblotting with an anti-KIF1C antibody. Clone #1 was selected for further assays. β-Actin provided a loading control. (C) KIF1C^KO cells were infected with viral particles encoding MSCV-driven KIF1C-BioID-3xFLAG plasmid to obtain near-endogenous KIF1C-BioID protein expression levels. Immunoblots were performed using the indicated antibodies. β-Actin provided a loading control. (D) A volcano plot showing enrichment versus significance of proteins identified in KIF1C-BioID experiments relative to control (BioID alone) experiments. Proteins not present in the BioID control or with an enrichment ratio greater than threefold and a P value >0.05 relative to the control (dashed red lines) were considered significant hits. KIF1C, Hook3, and Tc-Tex-1 (DYNLT1, a dynein light chain) are marked in red. (E) Immunoprecipitation (IP) of endogenous Hook3 and KIF1C with the indicated antibodies from 293T cells. Immunoblots were performed with anti-Hook3 or KIF1C antibodies. (F) Human Hook1, Hook2, and Hook3 tagged with the HaloTag on their amino termini and 3xFLAG on their carboxy termini were transiently transfected into 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. (G) Human KIF1A, KIF1B, KIF1C, KIF5A, KIF5B, and KIF5C were each tagged with BioID-3xFLAG on their carboxy termini and stably expressed in 293T cells. Tagged proteins were immunoprecipitated with FLAG affinity resin (FLAG-IP), and immunoblots were performed with anti-Hook3 and anti-FLAG antibodies. BioID-3xFLAG provided a control. Protein molecular weight markers are shown in kilodaltons on the anti-FLAG immunoblot.