Extended Data Fig. 2: The generation of CrK66- and AcK66-EB1 proteins by unnatural amino acids (UAAs) incorporation method in E.coli.
From: Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis
(a) Schematic diagram showing the expression of CrK66-EB1 and AcK66-EB1 in E. coli. The UAG in the mRNA of EB1 was decoded by tRNACUA in the presence of N-ε-crotonyl-lysine or N-ε-acetyl-lysine, giving rise to CrK66-EB1 or AcK66-EB1. NAM (10 mM) was added to prevent decrotonylation or deacetylation. (b) Diagram of the plasmid construction used to express recombinant CrK66/AcK66 and wild-type EB1 in E.coli. (c) Validation of AcK66-EB1 and CrK66-EB1 proteins generated as in b via CBB staining. Purified wild-type, AcK66- and CrK66-EB1 were also analyzed by AcK66-EB1 and CrK66-EB1 antibodies. (d) The specificity of CrK66-EB1 and AcK66-EB1 antibodies and the EB1 knockout efficiency were detected in CRISPR/Cas9-mediated EB1 knockout HeLa cells. Doxycycline (Dox, 1 μg/mL) was added to induce EB1 knockout for indicated time. Cell lysates were probed with anti-CrK66-EB1, AcK66-EB1 and EB1 antibodies. (e) HeLa cells depleted of endogenous EB1 were fixed and stained with CrK66-EB1, EB1 and α-tubulin antibodies. DNA was stained with DAPI. Scale bar, 10 μm. (f) HeLa cells were synchronized to the indicated time points by double thymidine release. Whole cell lysates (WCL) were probed for Lys66 crotonylation and acetylation levels by Western blotting with indicated antibodies. (g-h) Quantification of CrK66-EB1 and AcK66-EB1 levels in synchronized HeLa cells at G1/S or mitosis. Purified CrK66-EB1 and AcK66-EB1 proteins from E. coli were used as standard. Related to Fig. 1.
