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. 2021 Feb 5;220(4):e202007033. doi: 10.1083/jcb.202007033

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© 2021 Ayukawa et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S1. — Preparation of Drosophila tubulin. (A) SDS PAGE showing each step in preparation of WT tubulin. Lanes: (1) Cell lysate. (2) Diethylaminoethyl sepharose anion exchange column eluent. Red and black open arrowheads, recombinant α- and β-tubulin; red and black filled arrowheads, endogenous α- and β-tubulin, respectively. (3) His-tag affinity column eluent. (4) FLAG-tag affinity column eluent. (5) Final product after tag cleavage. (B) Analysis of nucleotide contents by ion exchange chromatography. The percentage indicates the occupancies at the E-site calculated from each chromatogram, assuming equal numbers of exchangeable and nonexchangeable nucleotide sites per tubulin dimer and that the nonexchangeable nucleotide is GTP. mAU, milli-absorbance unit. (C) CryoEM images of WT and Y222F MTs.