taxol stabilized microtubules  (Cytoskeleton Inc)

Journal: bioRxiv

Article Title: N-terminal phosphorylation inhibits Arabidopsis katanin and affects vegetative and reproductive development in opposite ways

doi: 10.64898/2026.01.20.700631

Figure Lengend Snippet: The KTN1 DDD version has decreased microtubule severing activity in vitro . (A) Diagram showing the location of the three experimentally validated serine phosphorylation sites in Arabidopsis KTN1. (B-E) Time course of rhodamine-labeled, taxol-stabilized microtubules incubated with 2mM ATP and 25nM of the indicated p60 katanin versions. See also Supplementary Videos 1-3. (B) KTN1 (n = 6), phosphomimetic and phosphonull mutants of either S92 (n = 5 each), S147 (n = 6 each), or S199 (n = 5 each). (C) KTN1 (n = 6) and double phosphomimetic mutations at S92 and S147 (n = 6), S92 and S199 (n = 6 movies) and S147 and S199 (n = 5). (D) KTN1 (n = 16), triple phosphonull (AAA, n = 15), and triple phosphomimetic (DDD, n = 13) mutant. (E) Extended duration of the DDD time course shown in (D) . Scale bar = 10 µm. ( F, G ) Plots of microtubule fluorescence signal over time from experiments in (C) and (D) . Each image in a series is normalized to the fluorescence signal of the first frame of that series. Error bars represent SEM of at least three separate protein preparations.

Article Snippet: The ATPase activity of 25 nM of KTN1, DDD, or AAA in the presence of either 25nM or 250nM of taxol-stabilized microtubules was evaluated using a commercially available ATPase ELIPA Kit (Cytoskeleton #BK051) according to the manufacturer’s instructions.

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Labeling, Incubation, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: N-terminal phosphorylation inhibits Arabidopsis katanin and affects vegetative and reproductive development in opposite ways

doi: 10.64898/2026.01.20.700631

Figure Lengend Snippet: DDD has lower microtubule binding affinity and ATPase activity in vitro . (A) Representative Coomassie Blue-stained SDS-PAGE gels of microtubule co-sedimentation assays. 1 µM KTN1 (n = 3), DDD (n = 3), or AAA (n = 6) was incubated with the indicated concentration of taxol-stabilized microtubules. S, supernatant; P, pellet. Arrows identify the different protein bands. (B) Binding curves of KTN1, DDD, and AAA proteins corresponding to (A) . Each data point represents the mean ± SEM. Data were fit to a one-site saturation binding model to obtain the microtubule binding affinity and maximum amount of protein binding. (C-E) Plots of ATPase activity over time of 25 nM of KTN1 (C) , DDD (D) , and AAA (E) proteins. ATPase activity was measured as the amount of Pi release in the absence (black) or presence of either 25 nM (blue) or 250 nM microtubules (red). Each data point represents the mean ± SEM from 9 independent experiments.

Article Snippet: The ATPase activity of 25 nM of KTN1, DDD, or AAA in the presence of either 25nM or 250nM of taxol-stabilized microtubules was evaluated using a commercially available ATPase ELIPA Kit (Cytoskeleton #BK051) according to the manufacturer’s instructions.

Techniques: Binding Assay, Activity Assay, In Vitro, Staining, SDS Page, Sedimentation, Incubation, Concentration Assay, Protein Binding

Journal: bioRxiv

Article Title: Integrative Genomic and Functional Analyses Reveal NINL as a Modulator of Tau Aggregation

doi: 10.64898/2025.12.12.694063

Figure Lengend Snippet: HEK293T cells were transiently transfected with pCMV6 NINL-myc plasmid or vector control plasmid for 48 hours. A. Schematic of the components of the autophagy-lysosome pathway that were evaluated. B. qPCR for NINL RNA. **, p = 0.0022. Student’s t-test. C. LysoTracker Median Fluorescence Intensity (MFI) quantified by flow cytometry plotted relative to vector control. ***, p = 0.0001. Student’s t-test. D. Representative immunoblot for Nlp ( NINL ), Lamp1, LC3B, and B-actin. E. Immunoblot quantification of Lamp1 protein levels normalized to vector controls. Ns, not significant. Student’s t-test. F. Immunoblot quantification of LC3B II/I ratio normalized to vector controls. *, p = 0.0224. Student’s t-test. G. Representative images from live imaging with CytoID. Both vector and NINL expressing cells were treated with rapamycin and chloroquine for 6 hours prior to imaging as a positive control. CytoID (autophagic vesicles) in green, Hoescht (nuclie) in blue, and ViaFluor (cytoskeleton) in red. Scale bar, 10μm. H. Quantification of the number of CytoID puncta per cell, normalized to the cell size (34-80 cells quantified per condition). ***, p = 0.0002; ****, p < 0.0001. Two-way ANOVA. I. Quantification of the average CytoID puncta size per cell. ****, p < 0.0001; **, p = 0.0048; *, p = 0.0151. Two-way ANOVA. Graphs represent mean ± SEM. Results represent 3 independent experiments with 2-3 replicates per condition in each experiment.

Article Snippet: Two hours prior to imaging, cells were treated with ViaFluor Live Cell Microtubule Stain (Biotium, Cat #: 70063), and Verapamil HCl (Enzo, Cat #: ENZ-51031) was added at a final concentration of 10μm according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Control, Fluorescence, Flow Cytometry, Western Blot, Imaging, Expressing, Positive Control