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. Author manuscript; available in PMC: 2016 Apr 3.
Published in final edited form as: Biochem Biophys Res Commun. 2015 Feb 26;459(2):259–263. doi: 10.1016/j.bbrc.2015.02.095

Fig. 2.

Fig. 2

Autoprocessing of chimera TxB-Ar. (A) InsP6 induced autoprocessing of TcdB and TxB-Ar. 10 ng/μL of TcdB or TxB-Ar was incubated in the reaction buffer containing 25 μM InsP6 at 37 °C for the indicated time. The reaction was terminated by SDS-sampling buffer and heating at 95 °C for 5 min. A VHH antibody (E3) against the GTD of TcdB was used for western blot analysis. (B) CPD conformational change probed by fluorescent AWP19. TcdB (0.1 μg/μL) was incubated with the indicated doses of AWP19 in 20 μM pH8.0 Tris buffer containing 25 μM InsP6 at 37 °C for 1 hour. The reactions were stopped by SDS sampling buffer and the samples were loaded on a SDS-PAGE. Fluorescence was measured using G-Box Chemi system and the total proteins were visualized on the gel after coomassie blue staining. The arrow indicates the cleaved GTD fragment.