1. 1234567891011 His-eIF5A Lys Elution 50 20 FTL Online Resource Figure 1. Purification of 6xHis-eIF5A Lys from E. coli. E. coli strain M15 was transformed with pQE-eIF5A and production of the recombinant yeast protein 6xHis-eIF5A Lys was obtained after induction of expression. The cell pellet was washed and suspended in ice-cold lysis buffer. The 6xHis-eIF5A Lys fusion protein was purified by affinity chromatography on Ni-NTA resin and fractions were analyzed by SDS-PAGE with Coomassie staining. Lanes 2 and 3 contain the flowthrough and the washing fractions. The 6xHis- eIF5A Lys was eluted in fractions 4 to 12. The purified protein from lanes 7 to 11 were used in Figure 1. eIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer Camila Arnaldo Olhê Dias a, Wanius Garcia b, Cleslei Fernando Zanelli a, Sandro Roberto Valentini a, * a Department of Biological Sciences, School of Pharmaceutical Sciences, UNESP – Univ Estadual Paulista, Araraquara, SP, Brazil b Center of Natural Sciences and Humanities, UFABC – Univ Federal do ABC, Santo André, SP, Brazil. * Corresponding autho: Faculdade de Ciências Farmacêuticas – UNESP, Rodovia Araraquara-Jaú, km 01, Araraquara, SP, Brazil, 14801-902, Telephone: +55 16 3301-6954, Fax: +55 16 3301-6940, E-mail: valentsr@fcfar.unesp.br 12

2. Online Resource Figure 2. SDS-PAGE of eIF5A proteins purified from bacteria and yeast. The proteins purified by ion exchange (bacteria) or affinity chromatography (yeast) were analyzed by SDS-PAGE with Coomassie staining. Lane 2 shows the recombinant yeast eIF5A Hyp purified from bacteria. Lanes 3 and 4 contain the proteins 6xHis-eIF5A Hyp and 6xHis-eIF5A K51R purified from yeast. These proteins were used in Figures 1 and 2. His-eIF5A K51R purified from yeast eIF5A Hyp purified from bacteria 1234 50 20 His-eIF5A Hyp purified from yeast

3. Online Resource Figure 3. Absorption spectra of purified proteins. (a) Recombinant yeast 6xHis-eIF5A Lys protein purified from bacteria and used in Figure 1. (b) Recombinant yeast eIF5A Hyp protein purified from bacteria and used in Figure 1. (c) Recombinant yeast eIF5A Lys protein purified from bacteria for the GST- pulldown in vitro assay (Figure 3c). a b c