Dhx36

Cells were washed with ice-cold PBS and lysed in ice-cold RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 10 mM Tris–HCl pH 8.0) supplemented with phosphatase and protease inhibitor cocktails (Sigma-Aldrich). The lysates were incubated on ice for 30 min, with occasional vortexing, and centrifuged at 12,000 g for 10 min at 4 °C. The supernatants were transferred and assayed for protein concentration using a Quick Start Bradford protein assay kit (Bio-Rad). Equal amounts of proteins (20–80 μg) were subjected to SDS–PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad). Antibodies to the following proteins were used: PABPC1 (Cell Signaling Technology), LARP1 (Cell Signaling Technology), PAIP1 (Thermo Scientific), PAIP2 (Santa Cruz Biotechnology), RPL29 (Thermo Scientific), PUS7 (Sigma-Aldrich), DHX36 (Protein Tech), FLAG (Sigma-Aldrich) and puromycin (Merck Millipore). Either β-actin (Sigma-Aldrich) or GAPDH (Abcam) were used as a loading control. The antibody dilutions can be found in the Reporting Summary.

Full table and dilutions for each antibody used can be found in Supplementary Table 4. YB-1 (Abcam: Ab12148), G3BP1 (Santa Cruz: sc81940), TIA1 (Santa Cruz: sc-1751), TIAR (Santa Cruz: sc-1749), eIF4G (Santa Cruz: sc-11373), Nucleolin (Santa Cruz: sc-9893), eIF4E (Santa Cruz: sc9976), Fus/TLS (Protein Tech Group: 11570-1-AP), TDP43 (Protein Tech Group: 10782-2-AP), Vigilin (Santa Cruz: 2404C3a), DHX36 (Protein Tech Group: 13159-1-AP), and GRSF1 (Aviva: ARP40382-P050).