Fig. 7: Systematic evaluation of β-tubulin residues that mediate taccalonolide binding.

a The key tubulin residues that mediate the binding affinity of 12 in the docking model structure generated by CovDock. The residues within the radius of 3.5 Å from the probe are displayed in the left graph and hydrophobic interactions are depicted by green dashed lines in the right graph. b, c HeLa cells were transfected with GFP-tagged TUBB1 constructs with indicated mutations then treated with 1 µM 11 for 8 h. Probe-treated cells were lysed and subjected to immunoblotting. b An anti-fluorescein antibody was used to detect 11 bound to endogenous β-tubulin (lower band, 50 kDa) and the GFP-tubulin constructs (upper band, 77 kDa). c β-tubulin immunoblotting showed the expression of the GFP-tubulin constructs (upper band, 77 kDa) and endogenous tubulin (lower band, 50 kDa). d, e Ratio of the binding of 11 to each ectopically expressed tubulin mutant d or endogenously expressed tubulin e normalized to wild type (WT). Data are shown as average ± SEM for n = 3 independent experiments other than WT and D226A, which are from n = 5 independent experiments. One-way ANOVA and Tukey’s post-hoc test were used to calculate statistical significance between each condition. Significance as compared to the R278A mutant that did not impact binding but was not used in data normalization is shown: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.