Fig. 4: eIF4B Ser93 phosphorylation boosts mesenchymal markers translation by enhancing eIF4B translational activity and stability.

A Western blotting of EMT markers (Vim, Snai1, and E-cad) in NC and eIF4B knockdown cells. B The levels of mesenchymal mRNA (N-cad, Vim, Snai1 and FN1) were measured by RT‒qPCR. C RT-qPCR analysis of the mRNA abundance of mesenchymal markers in polyribosome fractions collected by sucrose gradient centrifugation in shNC and sheIF4B CRC cells. D Cell lysates prepared from HT29 cells were subject to immunoprecipitation (IP) with antibodies against IgG and eIF4B, followed by RT-qPCR with primers specific for N-cad, Vim, Snai1 and FN1. Verification of eIF4B binding on N-cad, Vim, Snai1 and FN1 mRNA by agarose gel electrophoresis analysis of the qPCR products. The assays of (E–J) were performed in eIF4B WT, eIF4B S93A, and eIF4B S93D CRC cells, respectively. E Western blotting analysis of EMT markers. F The levels of mesenchymal mRNA were measured by RT‒qPCR. G RT-qPCR analysis of the mRNA abundance of mesenchymal markers in polyribosome fractions collected by sucrose gradient centrifugation. H The CHX pulse-chase assay of eIF4B was performed to detect the protein stability of eIF4B. I Protein synthesis assays showing relative levels of newly synthesized proteins. J The cells were treated with MG132 for 6 h, and then followed by ubiquitination assay performed with anti-eIF4B IP and anti-ubiquitin IB analyses. All the data are presented as mean±SEM from at least three independent experiments. P values were determined by two-sided Student t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.