x rhodamine  (Cytoskeleton Inc)

Journal: Developmental cell

Article Title: KIF2C promotes paclitaxel resistance by depolymerizing polyglutamylated microtubules.

doi: 10.1016/j.devcel.2025.03.004

Figure Lengend Snippet: Figure 5. 7S9 interrupts the MT depolymerization mediated by KIF2C (A) KIF2C activity was measured in the presence of 1 mM paclitaxel, 1 mM nocodazole, or 100 mM 7S9 as indicated. All data were normalized with the KIF2C activity measured in the presence of porcine brain tubulin and paclitaxel, represented as mean ± SEM of three replicates. (B) KIF2C pull-down assay was performed with porcine brain tubulin in the presence of indicated concentrations 7S9 (0–200 mM). (C) The in vitro MT depolymerization assay was performed with porcine brain tubulin in the presence of purified KIF2C (0.5 ng/mL), ATP (0.75 mM), and 7S9 (50 mM) as indicated. The heat map represents the fluorescence intensity of MTs. (D–F) 4T1 S1, R4, and R8 were treated with paclitaxel (1 mM), 7S9 (10 mM), or a combination of both, followed by immunofluorescence staining of tubulin (red) and DAPI (blue).

Article Snippet: In short, 400 nM recombinant kinesin protein was incubated with 300 nM microtubules in a 200 mL reaction buffer (1 mM paclitaxel, 75 mM KCl, 1 mM MgCl2, and 600 nM ATP) for 30 min at 37 C. The microtubules from porcine brain tubulin (Cytoskeleton) or purified from HEK293 cells in Free-style medium (293Fs) were dissolved in the BRB80 buffer (80 mM PIPES pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mMGTP, 1mMDTT, and 10%DMSO) at 37 C for 2 hr.

Techniques: Activity Assay, Pull Down Assay, In Vitro, Staining