Journal: eLife

Article Title: Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules

doi: 10.7554/eLife.44489

Figure Lengend Snippet: ( A ) List of mutations made in the wild type Ndc80 complex to generate cysteine-light construct. ( B ) Model of the Ndc80 complex using the dwarf tetramer structure (DOI: 10.2210/pdb5TCS/pdb) plus inserted coiled coil between the tetramerization domain and globular domain of each dimer. Break in coiled coil indicates estimated location of Ndc80 loop, indicated by black arrow. Colored balls on each globular domain of the model indicate location of the cysteine residues (Nuf2 S2C, Spc25 S154C) used for dual end-labeling. ( C ) ( Left ) Survival probability curves of residence times for Ndc80-GFP (data repeated from ) (black trace) and end-labeled Cy5-Ndc80-Cy3 (n = 1375) (pink trace). ( Right ) Bar graph of average residence times of the data at left. Error was calculated using bootstrapping analysis. p-Value was calculated using a two-tailed Student’s t test. Raw data of all residence times are included in . ( D ) Cartoon of approximate dye locations on the Ndc80 complex illustrating that in a more open conformation ( Top ), FRET would not occur. In a tightly bent conformation ( Bottom ), the dyes would be in close proximity and exhibit FRET. ( E ) Bulk FRET spectra of positive control double-stranded, dually-labeled oligonucleotide, negative control single-stranded singly-labeled oligonucleotides or end-labeled Ndc80 complex Nuf2 S2C Spc25 S154C. Displayed is the emission spectra of each construct from 560 nm to 730 nm under 550 nm (Cy3, donor) excitation. D em and A em denote the emission peaks of the donor (Cy3) and acceptor (Cy5). Values in parentheses indicate calculated relative FRET efficiency. Additional supplementary data are included in . 10.7554/eLife.44489.011 Figure 2—source data 1. Residence times on microtubules measured for individual, dual end-labeled Ndc80 complexes.

Article Snippet: Chemical compound , Cy5 maleimide , GE Healthcare Life Sciences , Cat#PA25031 , .

Techniques: Construct, End Labeling, Labeling, Two Tailed Test, Positive Control, Negative Control

Journal: eLife

Article Title: Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules

doi: 10.7554/eLife.44489

Figure Lengend Snippet: ( A ) Schematic of sequential end-labeling procotol for the Ndc80 complex used in bulk FRET assay. ( B ) ( Left ) Coomassie blue-stained gel showing end-labeled Ndc80 complex used in bulk FRET assay (SEC fraction shown as gray bar in elution profile to the right). Sequential overnight incubation with dyes leads to greater complex degradation, especially of the N-terminal tail on the Ndc80 protein, as seen in gel. ( Right ) Representative Superdex 200 16/60 elution profiles of end-labeled Ndc80 complex (Nuf2 S2C, Spc25 S154C) (black trace) used in bulk FRET assay normalized to highest peak. Gray bar shows SEC fractions collected. ( C ) Representative kymographs of Ndc80-GFP (black box) and Cy5-Ndc80-Cy3 (pink box).

Article Snippet: Chemical compound , Cy5 maleimide , GE Healthcare Life Sciences , Cat#PA25031 , .

Techniques: End Labeling, Staining, Labeling, Incubation

Journal: eLife

Article Title: Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules

doi: 10.7554/eLife.44489

Figure Lengend Snippet:

Article Snippet: Chemical compound , Cy5 maleimide , GE Healthcare Life Sciences , Cat#PA25031 , .

Techniques: Purification, Sequencing, Plasmid Preparation, Avidin-Biotin Assay, Software

Journal: Journal of Korean Neurosurgical Society

Article Title: In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model

doi: 10.3340/jkns.2017.0252

Figure Lengend Snippet: Surgical procedures for LV and CM injection. Surgical procedures for LV and CM injection were shown in (A) and (B), respectively. A : For LV injection, after anesthesia, rats were fixed with a stereotaxic device and cut to expose the bregma. After making a hole with a drill, a needle was injected into the LV. B : For CM injection, after anesthesia, rats were fixed with a stereotaxic device. After surgery to expose CM, Cy5.5 was injected into CM. C : Each region of interest to measure Cy5.5 was demonstrated. H : head, C : cervical, T : thoracic, L : lumbar, LV : lateral ventricle, CM : cisterna magna.

Article Snippet: 20 nM Cy5.5 (Amersham CyDye™ mono-reactive NHS Ester, GE Healthcare, Piscataway, NJ, USA) in 20 μL saline was injected using a syringe pump (LEGATO™111, KD scientific, Holliston, MA, USA).

Techniques: Injection

Journal: Journal of Korean Neurosurgical Society

Article Title: In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model

doi: 10.3340/jkns.2017.0252

Figure Lengend Snippet: Optical imaging of Cy5.5 after LV and CM injection. The total flux of Cy5.5 signal at each region of interest was evaluated at 12, 24, 48, and 72 hours after injection of Cy5.5 via LV and CM route. A : In the LV injection the signal of Cy5.5 was observed in the thoracic and lumbar region within 12 hours. B : In the CM injection, most Cy5.5 signal remained in the head region until 72 hours. LV : lateral ventricle, CM : cisterna magna.

Article Snippet: 20 nM Cy5.5 (Amersham CyDye™ mono-reactive NHS Ester, GE Healthcare, Piscataway, NJ, USA) in 20 μL saline was injected using a syringe pump (LEGATO™111, KD scientific, Holliston, MA, USA).

Techniques: Optical Imaging, Injection

Journal: Journal of Korean Neurosurgical Society

Article Title: In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model

doi: 10.3340/jkns.2017.0252

Figure Lengend Snippet: Quantitation of the in vivo signal intensity of Cy5.5 after LV and CM injection. The total flux of Cy5.5 signal was quantitated at each region of interest. A : After LV injection, the sum of total fluxes was shown at each time point. B : The total flux of Cy5.5 at each region of interest was shown at various time points. C : After CM injection, the sum of total fluxes was shown at each time point. D : The total flux of Cy5.5 at each region of interest was shown at various time points. * p <0.05. LV : lateral ventricle, CM : cisterna magna.

Article Snippet: 20 nM Cy5.5 (Amersham CyDye™ mono-reactive NHS Ester, GE Healthcare, Piscataway, NJ, USA) in 20 μL saline was injected using a syringe pump (LEGATO™111, KD scientific, Holliston, MA, USA).

Techniques: Quantitation Assay, In Vivo, Injection

Journal: Journal of Korean Neurosurgical Society

Article Title: In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model

doi: 10.3340/jkns.2017.0252

Figure Lengend Snippet: Comparison of the in vivo signal intensity of Cy5.5 according to the injection route at each region of interest. The regional total flux of Cy5.5 at each time point was compared between LV and CM injection route. A : In head region, CM injection showed slightly higher signal intensity than that of the LV injection but not statistically significant. B : In cervical region, both injection methods showed similar signal intensity. C and D : In thoracic and lumbar region, LV injection showed significantly higher signal intensity than CM injection. * p <0.05. LV : lateral ventricle, CM : cisterna magna.

Article Snippet: 20 nM Cy5.5 (Amersham CyDye™ mono-reactive NHS Ester, GE Healthcare, Piscataway, NJ, USA) in 20 μL saline was injected using a syringe pump (LEGATO™111, KD scientific, Holliston, MA, USA).

Techniques: In Vivo, Injection