Superdex s200

An IgG4 isotype antibody was labeled with Sulfo‐Cyanine‐7.5 dye using NHS ester chemistry (66320, Lumiprobe). The antibody was prepared at ≈10 mg mL⁻¹ in 90 mm carbonate, 9 mm phosphate buffer with 125 mm sodium chloride at pH 8.3. The dye was dissolved in a one‐tenth volume of carbonate buffer immediately before adding to the antibody at a 10 equivalent excess and incubated at 25 °C for 4 h. The labeled antibody was isolated from the excess dye on a size exclusion chromatography column (Superdex S200, Cytiva) with a mobile phase of 1× PBS, pH 7.2 at 1 mL min⁻¹. The degree of labeling was determined using MALDI‐MS to be ≈4.5 dyes per antibody. The antibody was concentrated to ≈30 mg mL⁻¹ using a 15 mL spin concentrator (Millipore) with a 100 kDa MWCO membrane. Samples of labeled and unlabeled antibody (5–10 µg) were run on an HPLC (Agilent 1260 Infinity II) using an Agilent AdvanceBio SEC 300Å 2.7 mm column (PL1580‐3301, Agilent) at 1 mL min⁻¹ in a mobile phase of 1× PBS; pH 7.2 (20012‐027, GIBCO) with peaks detected by absorbance at 214 nm. The total run time was 7 min.

Enzymes: Ulp1-protease (recombinant, purified in-house), recombinant 4E-BP1 (Sino Biological, 10022-H07E), BamHI-HF (R3136, NEB UK), BsaI-HF (R3535, NEB UK), NotI-HF (R3189S, NEB UK), HindIII-HF (R3104S, NEB UK), NcoI-HF (R3193, NEB UK), NsiI-HF (R3127, NEB UK). Antibodies: eIF4A1 (ab31217, Abcam UK), GFP (ab13970, Abcam UK), vinculin (ab129002, Abcam UK). Kits: HiScribe™ T7 ARCA mRNA Kit (NEB E2065S), HiScribe™ T7 mRNA Kit with CleanCap® Reagent AG (NEB E2080S), Rabbit Reticulocyte Lysate (Promega L4151), TMT 16plex reagent kit (A44522 Thermo Scientific). RNAs: all RNAs were purchased from IBA life sciences or IDT, see Supplementary Table S8. Columns for FPLC: HisTrap 5 ml (17524802, Cytiva), ResourceQ 6 ml (17117901, Cytiva), Heparin 5 ml (17040701, Cytiva), Superdex S200 16/60 (28989335, Cytiva), Superdex S200 increase 3.2/300 (28990946, Cytiva). eIF4A-inhibitors: silvestrol (Generon UK), hippuristanol (gift from John Le Quesne). SILAC amino acids: Lys-¹²C₆¹⁴N₂ (Lys0), Arg-¹²C₆¹⁴N₄ (Arg0), Lys-¹³C₆¹⁵N₂ (Lys8), Arg-¹³C₆¹⁵N₄ (Arg10) all purchased from Cambridge Isotope Laboratories (#ULM-8766, #ULM-8347, #CNLM-291-H, #CNLM-539-H)