Fig. 6: The taccalonolide epoxide and β-tubulin D226 are critical for covalent binding. | Nature Communications

Fig. 6: The taccalonolide epoxide and β-tubulin D226 are critical for covalent binding.

From: Elucidating target specificity of the taccalonolide covalent microtubule stabilizers employing a combinatorial chemical approach

Fig. 6

a HCC1937 cells were treated with 0.05–5 µM taccalonolide probes with (11) or without (10) the 22,23-epoxide for 24 h. Colocalization of taccalonolide probes (green) with β-tubulin immunofluorescence (red) was evaluated by confocal imaging. b, c HCC1937 cells treated with 5 µM 10 or 11 for 6 h were lysed and subjected to immunoblotting using an anti-β-tubulin antibody b or an anti-fluorescein antibody c. d, e HeLa cells were transfected with GFP-tagged TUBB1 constructs with indicated mutations then treated with 1 µM 11 for 8 h. Cell lysates were harvested for immunoblotting. β-tubulin immunoblotting d showed the expression of endogenous tubulin (50 kDa) and the GFP-tubulin constructs (77 kDa). e An anti-fluorescein antibody was used to detect 11 bound to endogenously expressed β-tubulin (lower band, 50 kDa) and the GFP-tubulin constructs (upper band, 77 kDa). Scale bars = 10 µm for all images. Source data are provided as a Source Data file.

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