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. 2022 Nov 2;13:6558. doi: 10.1038/s41467-022-34052-8

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© The Author(s) 2022

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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Fig. 3 — a Parental mESC and double knock-in Halo-eIF4E^+/+/SNAP-eIF4G^+/+ total cell lysates (input) were subjected to cap-pull-down assay (m⁷GTP-pull-down) and analyzed by western blotting. eIF4G and eIF4E antibodies showed binding of wild-type and tagged proteins to the 5’cap. Tagging of endogenous eIF4E and eIF4G did not affect cap-binding as compared to the parental counterpart. b–e Halo-eF4E^+/+/SNAP-eIF4G^+/+ cells were treated with vehicle (DMSO) or 250 nM torin-1 for 2 h and 5 h respectively. b Simultaneous diffusion of _JF585Halo-eIF4E and _JF646SNAP_f-eIF4G was analyzed by dual color cross-correlation spectroscopy (FCCS) in the indicated conditions. Cross-correlation was detected in the cytoplasm of control cells (left panel), but not in the nucleus (right panel). Minor residual eIF4E:eIF4G was detected in the cytoplasm upon 2 h mTOR inhibition (left panel). c, d Averaged autocorrelation curves representing individual diffusion of _JF585Halo-eIF4E (c) and _JF646SNAP_f-eIF4G (d) in the indicated conditions (N = 10 ± SEM). In control cells, both _JF585Halo-eIF4E (c) and _JF646SNAP_f-eIF4G (d) autocorrelations showed slower diffusion as compared to their nuclear counterparts. No changes were detected in the nuclear counterparts. Cytoplasmic eIF4E molecules diffuse as fast as the nuclear counterpart as early as 2 h upon torin-1 treatment, whereas cytoplasmic eIF4G mirrors the nuclear diffusion only after 5 h. e Total lysates (input) of cells described in (c–e) were subjected to cap-pull-down assay (m⁷GTP pull-down) and analyzed by western blotting with the indicated antibodies. eIF4E:eIF4G dissociation occurred as early as 2 h upon torin-1 treatment.