Sc 9976

The proximity ligation assay was performed with the kit from Sigma—Duolink™ In Situ Orange Starter Kit Mouse/Rabbit (Ref: DUO92102) with λ_ex 554 nm; λ_em 576 nm (Cyanine 3; Zeiss Filter set 20) according to the manufacturer's recommendations. The Nunc™ Lab-Tek II Chamber Slide (Thermo Scientific Nunc, Ref: 154534) was used for the proximity ligation assay. The primary antibodies used were: rabbit anti-UPF1 antibody at 1:250 (Abcam ab86057), rabbit anti-UPF2 antibody at 1:250 (Eurogentec), rabbit anti-UPF3X antibody at 1:250 (Abcam ab134566), mouse anti-AKT1 antibody at 1:50 (Santa Cruz sc-271149), mouse anti-eIF4E antibody at 1:250 (Santa Cruz sc-9976), rabbit anti-MDM2 antibody at 1:250 (Cell Signaling #86934).

Cells were lysed in lysis buffer (25 mM Tris pH 8.0, 125 mM NaCl, 1 mM MgCl₂, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1X protease inhibitor (Sigma-Aldrich), phosphatase inhibitor cocktails #2 and #3 (Sigma-Aldrich), and 1 mM PMSF). Cell lysates were resolved on a Nupage 4–12% Bis-Tris gradient gel (Life Technologies) and probed using antibodies recognizing phospho- and total eIF4E1 (Cell Signaling #9741 and Santa Cruz sc-9976), phospho- and total ERK (#9101 and #9102, Cell Signaling), c-Myc (#9402), n-Myc (#9405), p-MNK1/2 (#2111), dicer (#3363), eIF4A (#2013) (Cell Signaling), GAPDH (ab8245), BTK (ab54219), YY1 (ab12132) (Abcam), eIF4E3 (Proteintech: N-terminal, #17282-1-AP), MNK1 (sc-6965), MNK2 (sc-6964), CDK6 (sc-7961), eIF4G (sc-11373), MCL-1 (#sc-819) (Santa Cruz) or GFP (EVN-AB513, Axxora). All primary antibodies were used at 1:1,000 dilution. Densitometry analyses were performed using ImageJ software (NIH) and presented as ratio of target band signal intensity to GAPDH band signal intensity. Full immunoblots are presented in Supplementary Fig. 10.