Tubulin can be directly arginylated by ATE1 in vitro and in cells. (A) Independent repeat of the result from Fig. 1 A. Time course in vitro arginylation reaction showing Coomassie blue (top) and autoradiography (bottom). (B) In vitro arginylation reactions on tubulin and/or ⍺-synuclein with Coomassie blue (top) and autoradiography (bottom) to show that incorporation of radiolabeled Arg into the 55 kDa band is specific to tubulin reactions, as opposed to being a contaminant in or degradation product of purified ATE1 or RRS. + indicates a full reaction, and – indicates a negative control. (C) Independent repeat of the result from Fig. 1 B. In vitro arginylation on soluble tubulin and polymerized microtubules showing Coomassie blue (top) and autoradiography (bottom). (D) Coomassie blue gel showing the polymerization status of tubulin used for the in vitro arginylation reactions in Fig. 1 B. Most of the tubulin sample is in the supernatant, and most of the microtubule sample is in the pellet. (E) In vitro arginylation reactions on microtubules polymerized with the GMPPCP, a non-hydrolyzable GTP analog, with Coomassie blue (top) and autoradiography (bottom) to show that arginylation of microtubules is not an artifact of Taxol treatment. (F and G) Representative spectra of E77 arginylation on ⍺-tubulin (F) and D74 arginylation on β-tubulin (G) from Taxol-purified microtubules from wild-type MEFs. Source data are available for this figure: SourceData FS1.