Fig. 2: Characterization of αE254Q-microtubules.
From: Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap
A Coomassie-stained SDS gel of purified recombinant human αE254Q-tubulin (right lane). The quality of the purified protein in the gel is representative of three independent purifications (N = 3) from separate batches of αE254Q-tubulin-expressing insect cells (for original purification gels see the Source Data file). B Example HPLC chromatograms of nucleotides extracted from microtubules composed of WT (green), αE254A (red), and αE254Q (blue) recombinant tubulin. Dark and light gray boxes highlight the areas of the GDP peak (elution time ≈ 8.4 min) and the GTP peak (elution time = 11.6 min), respectively. C Bar graph showing the mean nucleotide composition of WT, αE254A, and αE254Q-microtubules. Dark and light gray bars represent the average percentages of GDP and GTP, respectively. N = 2 independent experiments; n = 2 replicas for each condition. Error bars represent SEM. D Overlaid IRM and TIRF microscopy images of αE254Q-microtubules (black) polymerized for 20 min from glass-attached GMPCPP microtubule seeds (black) in the presence of 40 nM mGFP-EB3 (green). N = 4 independent experiments. E Example IRM/TIRF microscopy kymographs of WT and αE254Q-microtubules. F TIRF microscopy images of αE254Q-microtubules in the presence of different concentrations of mGFP-EB3 (green). αE254Q-microtubules were pre-grown in solution from CF640R-labeled “GMPCPP-seeds” (purple) and then surface-immobilized, followed by the addition of mGFP-EB3. G Plot of average maximum mGFP-EB3 fluorescence intensities measured along αE254Q-microtubules at the different EB3 concentrations indicated in (F). A hyperbolic fit (black line) yields the dissociation constant, Kd, as indicated. N = 2 independent experiments; n = 100 microtubules per each mGFP-EB3 concentration. Error bars represent the SEM. Fluorescence intensities are represented in arbitrary units (A.U.). White and black horizontal scale bars represent 10 μm; vertical scale bar represents 5 min. Source data are provided as a Source Data file.
