Sc 11373

Cells were lysed with NP-40 lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40) containing 1 mM PMSF (Sigma) and centrifuged to remove debris. Protein concentration in supernatant was assayed using a Quick Start Bradford Protein Assay reagent (Bio-Rad). Each sample containing 90 μg of protein was resolved on 3–20% SDS-PAGE. Upon transfer, Western blot was done with mouse monoclonal anti-utrophin (MANCHO03 clone 8A4, developed by Glenn E. Morris and obtained from the Developmental Studies Hybridoma Bank at the University of Iowa), rabbit anti-eIF4G (sc-11373, Santa Cruz Biotechnology) and mouse anti-β-actin (sc-81178, Santa Cruz Biotechnology) antibodies.

1×10⁵ U2OS cells were plated into a 24-well dish seed with coverslips. The following day cells were treated as indicated in figure legends. Then the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with −20°C methanol for 5 min, and blocked for at least 20 min with 5% normal horse serum (NHS; ThermoFisher) diluted in PBS. Primary antibodies were diluted in blocking solution and incubated for 1 h at room temperature or overnight at 4°C. Antibodies against G3BP1(sc-365338), eIF4G (sc-11373), eIF3b (sc-16377), FMRP (sc-101048) FXR1 (sc-10554), TIAR (sc-1749), Hedls (sc-8418) and HuR(sc-5261) were all purchased from Santa Cruz and used at a 1:250 dilution. The DDX6 antibody was purchased from Bethyl Laboratories (A300-461A) and used at a 1:500 dilution. Coverslips were washed three times for 5 min then incubated with secondary antibodies and Hoechst 33258 (Sigma-Aldrich) for 1 h at room temperature and again washed. Coverslips were mounted on glass slides with Vinol.