Fig 2: Co-localization of the eIF4E isoforms with PBs and SGs during heat shock. The eIF4E1, 2, 3 proteins (green) were ectopically produced in fusion with GFP in U2OS cells. Nineteen hours after transfection, the cells were exposed to 41.7 °C for 30 min, fixed and assessed for eIF3B-stained SGs (red) and DDX6-stained PBs (blue). Co-localization of the particular eIF4E with SGs and PBs is demonstrated in the boxed area replicated in higher magnification on the right side of each panel and by the intensity profile measured along the dashed white line within the boxed area. Both eIF4E1 (a, b) and all three eIF4E2 (c– e) variants co-localized with SGs and PBs. The eIF4E3_A (f) was recruited only to SGs, and eIF4E3_B (g) co-localized with neither SGs nor PBs. Approximately 50 cells transfected with either vector were observed in two independent biological replicates. Scale bar, 20 µm

Fig 3: eIF4G interacts with eIF4E3_A but not with eIF4E3_B. a Coomassie blue stained gel demonstrating immunoprecipitation of the ectopically expressed GFP-eIF4E3_A from the HEK293 cell lysate using a GFP-Trap approach. M PageRuler™ Prestained Ladder (Thermo Scientific); INPUT whole cell lysate; eIF4E3_A-IP proteins co-immunoprecipitating with GFP-eIF4E3_A. b MS analysis of the proteins co-immunoprecipitating with eIF4E3_A. Gel slices are numbered as in panel A. c Western blots of proteins co-immunoprecipitating with eIF4E1_1, eIF4E3_A and eIF4E3_B transiently expressed in GFP fusion in HEK293 cells using GFP-Trap agarose beads (GFP-TRAP). Membranes were developed with anti-GFP (detecting eIF4E-GFP fusion proteins), anti-eIF4G and anti-ß-actin antibodies. INPUT lines including ß-actin and eIF4G served as a loading control. Lysate from non-transfected HEK293 cells was used as a negative control (mock). The absence of detectable ß-actin on GFP-Trap beads shows no contamination of non-specifically bound proteins in the samples

Fig 4: Co-localization of the eIF4E proteins and their isoforms with PBs and SGs during oxidative stress. The eIF4E1, 2, 3 proteins (green) were ectopically produced in fusion with GFP in U2OS cells. Nineteen hours post-transfection, the cells were treated with 1 mM sodium arsenite for 40 min, fixed and assessed for eIF3B-stained SGs (red) and DDX6-stained PBs (blue). Co-localization of the particular eIF4E with SGs and PBs is demonstrated in the boxed area on the right side of each panel and by the intensity profile measured along the dashed white line within the boxed area. Contrary to heat shock, only the eIF4E1 variants were able to co-localize with both SGs and PBs (a, b). The eIF4E2 protein variants (c–e) co-localized only with PBs. eIF4E3_A (f) was present only in SGs, and eIF4E3_B (g) co-localized with neither SGs nor PBs. Approximately 50 cells transfected with either vector were observed in two independent biological replicates. Scale bar, 20 µm

Fig 5: GFP-eIF4E1 fusion protein is capable to bind to m7GTP agarose. HEK293 cells were lysed (INPUT) and the lysate was incubated with the m7GTP-agarose. Western blot was developed with anti-eIF4E1 antibody, which clearly shows that both endogenous eIF4E1 and its GFP-eIF4E1 fusion counterpart retain their ability to bind the m7G cap. Actin was used as a control of a sufficient washing of the m7GTP affinity resin