Fig. 2: HURP contains tubulin-binding domains.
From: HURP binding to the vinca domain of β-tubulin accounts for cancer drug resistance
a Nickel pull-down assays of tubulin in the presence of his-tagged GFP-HURP FL, GFP-HURP (1-280) or GFP-HURP (200-620). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. b Tubulin pulled down by HURP in a was analyzed. The bar graph shows the fraction of tubulin bound. Data are mean ± SD from three independent experiments. Two-tailed Student’s t test; Statistical differences: **, P < 0.01; n.s., not significant. c, SEC (Superdex 200) elution profiles (arb. units) for the GFP-HURP (200-620)•tubulin complex (red line), GFP-HURP (200-620) (green line), and tubulin (blue line). The void volume (Vo) is indicated. d–f The peak fractions of the (d) GFP-HURP (200-620)•tubulin complex, (e) GFP-HURP (200-620), (f), and tubulin were analyzed by SDS-PAGE and stained with Coomassie blue. g, h The BLI analysis displayed the binding kinetics of (g) GFP-HURP FL or (h) GFP-HURP (200-620) (the ligand) to tubulin (the analyte) over time. Various concentrations of tubulin were assessed. Binding affinities are indicated. i SDS-PAGE analysis of microtubule cosedimentation assays in the presence of GFP-HURP FL, GFP-HURP (1-280) or GFP-HURP (200-620). Supernatant and pellet fractions are indicated as S and P, respectively. j, HURP proteins co-pelleting with the microtubules in i were analyzed. The bar graph shows the fraction of HURP proteins bound. Data are mean ± SD from three independent experiments. Two-tailed Student’s t test; Statistical differences: *, P < 0.05; n.s., not significant. k, l Representative kymographs showing microtubules grown from X-rhodamine-labeled GMPCPP-stabilized seeds with HiLyte FluorTM647 tubulin in the (k) absence or (l) presence of 500 nM GFP-HURP (200-620). Vertical scale bar = 90 sec, and horizontal scale bar = 10 μm. m–p Quantification of microtubule dynamicity, including (m) polyermization rate, (n) depolymerization rate, (o) catastrophe frequency and (p) microtubule length, as a function of the indicated GFP-HURP (200-620) concentrations. Data are mean ± SD from three independent experiments (n = 30 (m–o) and n = 24 (p) microtubules). Two-tailed Student’s t test; Statistical differences: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant.
