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Figure 5. Dynein and dynactin form an active motor complex with JIP3 in vitro, and doublecortin (DCX) reduces its velocity. (A) Illustrations of the JIP3 and DCX constructs (left) and the DDJ motor complex (right). (B) Kymographs of dynein in the absence and presence of dynactin, JIP3, DCX, and N-DCX. Dynein was labeled with SNAP-TMR (green) and JIP3 was labeled with Halo-JP646 (red). (C) The velocity of DDJ motor complexes, <t>KIF5B,</t> and gliding MTs powered by surface-absorbed single-headed dynein. The green bars represent the median with 95% CI. DDJ (DDJ only): 0.76 [0.65, 0.89]
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Figure 5. Dynein and dynactin form an active motor complex with JIP3 in vitro, and doublecortin (DCX) reduces its velocity. (A) Illustrations of the JIP3 and DCX constructs (left) and the DDJ motor complex (right). (B) Kymographs of dynein in the absence and presence of dynactin, JIP3, DCX, and N-DCX. Dynein was labeled with SNAP-TMR (green) and JIP3 was labeled with Halo-JP646 (red). (C) The velocity of DDJ motor complexes, <t>KIF5B,</t> and gliding MTs powered by surface-absorbed single-headed dynein. The green bars represent the median with 95% CI. DDJ (DDJ only): 0.76 [0.65, 0.89]
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Figure 5. Dynein and dynactin form an active motor complex with JIP3 in vitro, and doublecortin (DCX) reduces its velocity. (A) Illustrations of the JIP3 and DCX constructs (left) and the DDJ motor complex (right). (B) Kymographs of dynein in the absence and presence of dynactin, JIP3, DCX, and N-DCX. Dynein was labeled with SNAP-TMR (green) and JIP3 was labeled with Halo-JP646 (red). (C) The velocity of DDJ motor complexes, <t>KIF5B,</t> and gliding MTs powered by surface-absorbed single-headed dynein. The green bars represent the median with 95% CI. DDJ (DDJ only): 0.76 [0.65, 0.89]
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Addgene inc prescission dcx egfpstrepii plasmid addgene
Figure 5. Dynein and dynactin form an active motor complex with JIP3 in vitro, and doublecortin (DCX) reduces its velocity. (A) Illustrations of the JIP3 and DCX constructs (left) and the DDJ motor complex (right). (B) Kymographs of dynein in the absence and presence of dynactin, JIP3, DCX, and N-DCX. Dynein was labeled with SNAP-TMR (green) and JIP3 was labeled with Halo-JP646 (red). (C) The velocity of DDJ motor complexes, <t>KIF5B,</t> and gliding MTs powered by surface-absorbed single-headed dynein. The green bars represent the median with 95% CI. DDJ (DDJ only): 0.76 [0.65, 0.89]
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Figure 5. Dynein and dynactin form an active motor complex with JIP3 in vitro, and doublecortin (DCX) reduces its velocity. (A) Illustrations of the JIP3 and DCX constructs (left) and the DDJ motor complex (right). (B) Kymographs of dynein in the absence and presence of dynactin, JIP3, DCX, and N-DCX. Dynein was labeled with SNAP-TMR (green) and JIP3 was labeled with Halo-JP646 (red). (C) The velocity of DDJ motor complexes, KIF5B, and gliding MTs powered by surface-absorbed single-headed dynein. The green bars represent the median with 95% CI. DDJ (DDJ only): 0.76 [0.65, 0.89]

Journal: eLife

Article Title: Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking

doi: 10.7554/elife.82218

Figure Lengend Snippet: Figure 5. Dynein and dynactin form an active motor complex with JIP3 in vitro, and doublecortin (DCX) reduces its velocity. (A) Illustrations of the JIP3 and DCX constructs (left) and the DDJ motor complex (right). (B) Kymographs of dynein in the absence and presence of dynactin, JIP3, DCX, and N-DCX. Dynein was labeled with SNAP-TMR (green) and JIP3 was labeled with Halo-JP646 (red). (C) The velocity of DDJ motor complexes, KIF5B, and gliding MTs powered by surface-absorbed single-headed dynein. The green bars represent the median with 95% CI. DDJ (DDJ only): 0.76 [0.65, 0.89]

Article Snippet: DOI: https://doi.org/10.7554/eLife.82218 22 of 36 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- HA (mouse monoclonal) EMD Millipore 05- 904 (1 μg/ml dilution) Antibody Anti- GFP (mouse monoclonal) YenZym https://www.yenzym. com/default.html (0.1 mg/ml) Antibody Alexa 488- conjugated goat anti- rabbit IgG (H+L) (goat polyclonal) Thermo Fisher Scientific A11036 (1:10,000 dilution) Antibody Alexa 568- conjugated goat anti- rabbit IgG (H+L) (goat polyclonal) Thermo Fisher Scientific A- 11036 (1:10,000 dilution) Antibody Alexa 488- conjugated goat anti- mouse IgG (H+L) (goat polyclonal) Thermo Fisher Scientific A32723 (1:10,000 dilution) Antibody Alexa 568- conjugated goat anti- mouse IgG (H+L) (goat polyclonal) Thermo Fisher Scientific A- 11031 (1:10,000 dilution) Recombinant DNA reagent 6His- PreScission- DCX- EGFPStrepII (plasmid) Addgene #83918 Recombinant DNA reagent kif5b(1- 560)- EGFP- 6His (plasmid) Addgene #15219 Recombinant DNA reagent Sfp- 6His (plasmid) Addgene #75015 Recombinant DNA reagent Construct pGEX- 4T- 1 encoding JIP3 (plasmid) Gift from Dr. Valeria Cavalli; Sun et al., 2011 Recombinant DNA reagent Construct pCDNA3 encoding JIP3 (plasmid) Gift from Dr. Roger Davis; Kelkar et al., 2000 Recombinant DNA reagent pSNAP- tag(T7)2 (plasmid) NEB NEB #N9181S Recombinant DNA reagent pFastBac plasmid with codonoptimized full- length human dynein (plasmid) A gift from the Carter lab; Schlager et al., 2014 Recombinant DNA reagent The pFastBac plasmid encoding tail- truncated human dynein (amino acids 1320–4646) (plasmid) Gift from the ReckPeterson Lab; Htet et al., 2020 Recombinant DNA reagent Construct expressing IC- 1B (plasmid) Gift from Dr. Kevin Pfister; Ha et al., 2008 Recombinant DNA reagent Construct expression TrkB- RFP (plasmid) Gift from Dr. Xiaowei Zhuang Recombinant DNA reagent Plasmid pBA (plasmid) Gift from Dr. Gary Banker; Jacobson et al., 2006 Recombinant DNA reagent The pBA plasmid encoding HA- tagged N- DCX mutant (plasmid) This paper Available upon request ‘Materials and methods’ Recombinant DNA reagent The pBA plasmid encoding HA- tagged C- DCX mutant (plasmid) This paper Available upon request ‘Materials and methods’ Recombinant DNA reagent The pBA plasmid encoding HA- tagged DCX mutant T203R (plasmid) This paper Available upon request ‘Materials and methods’ Continued Continued on next page Fu et al. eLife 2022;11:e82218.

Techniques: In Vitro, Construct, Labeling