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. 2018 Apr 3;8:5545. doi: 10.1038/s41598-018-23845-x

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Preparation of cellular fractions. (A) Schematic of the procedure used to prepare cellular fractions S30, S100, R, RSW, and F from HEK293 cells. S30 extract is the total lysate obtained from cells. S30 ultracentrifugation yielded the S100 fraction (supernatant), and the ribosomes plus associated factors (R) (pellet). To prepare the fraction containing ribosomes free from associated factors (RSW), the ribosomal pellet was dissolved in high-salt buffer, loaded in a discontinuous sucrose gradient and ultracentrifuged. The supernatant of the ultracentrifugation yielded the F fraction. (B) HEK293 fractions corresponding to S30, S100, F (100 µg of total protein), ribosomes (R) and salt-washed ribosomes (RSW) (30 µg) were analyzed by Western blot on the same membrane to detect the presence of RACK1 (40 S subunit), the 60S ribosomal proteins P0 and P1/P2, the elongation factor eEF2, the initiation factors eIF4G, eIF4B, eIF4E, and eIF2α, and the IRES-interacting proteins PTB, Ebp1 and Gemin5. This figure shows horizontal slices of the WB carried out for each factor. Images of the un-cropped WB film obtained for each factor are shown in Supplementary Fig. S7).