Extended Data Fig. 5: The generation of CrK66- and AcK66-EB1 proteins by UAAs incorporation method in HeLa cells.
From: Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis
(a) Diagram of the plasmid construction used to express recombinant CrK66 or wild-type EB1 in HeLa cells depleted of endogenous EB1. (b) EB1-WT-GFP or EB1-K66TAG-GFP and pCMV-CrKRS/ acKRS were co-transfected into HeLa cells depleted endogenous EB1. CRISPR/Cas9-mediated endogenous EB1 knockout was achieved by addition of Doxycycline (Dox, 1 μg/mL) into CRISPR/Cas9-edited HeLa cells. The expression levels of EB1-GFP and CrK66-EB1 or AcK66-EB1 were analyzed by Western blotting with anti-GFP, anti-AcK66-EB1 and anti-CrK66-EB1 antibodies. (c) Schematic illustration of spindle positioning in mitotic HeLa cells expressing wild-type or Lys66-crotonylated EB1. Note that EB1 crotonylation at Lys66 causes slope of spindle in the z direction, which means that, when one spindle pole is just right on the focal plane, the other pole usually stays out of focus. Related to Fig. 2.
