Eef1a

Whole cell lysates were prepared from 10 mL of exponentially growing yeast in YEPD media using lysis buffer (50 mm Tris‐Cl, pH 7.5, 50 mm NaCl, 1% Triton X‐100, and 1 mm PMSF) and vortexing with glass beads. Fifty microgram of extract was incubated with 20 µL Protein A/G beads (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at 4 °C then centrifuged for 5 min to preclear the lysate. Lysates were then incubated with rabbit polyclonal antiserum to full length S. cerevisiae eEF1A (Kinzy Laboratory, Piscataway, NJ, USA) for 1 h at 4 °C, and the complex was precipitated by incubation with Protein A/G beads (1 h, 4 °C). Control reactions contained 10 µg of mouse IgG antibody (Millipore, Burlington, MA, USA). Immunoprecipitates were washed four times with lysis buffer, eluted using Laemmli buffer, and separated by SDS/PAGE. Membranes were blotted with rabbit polyclonal antiserum to full length S. cerevisiae eEF1Bα (Kinzy Laboratory) (1 : 5000) or eEF1A (1 : 10 000), and the secondary antibody (1 : 2000) was VeriBlot for IP Detection Reagent (Abcam, Cambridge, UK).

Antibodies used in western blot: Setdb1 (Abcam-ab107225), Vinculin (Sigma- V9131), alpha-tubulin (Sigma- T9026), GLP (R&D-PP-B0422-00), Trim71 (Fitzerald-70R-11-707), GFP (Santa Cruz-sc-9996), GM130 (BD-610822), eEF1A (Abcam-ab37969), Rpl5 (GeneTex- GTX101821), beta-actin (Sigma- A5441), GAPDH (Sigma-G9545), eIF3c (Novusbio-NB100-500), SDHA (CST-11998), Ago2 (Abcam- ab32381), c-Myc (clone 7E18, Sigma). Antibodies used for immunoprecipitation: Myc-Trap (ChromoteK-yta-20), GFP-Trap (ChromoteK-gta-20), IgG (Santa Cruz). Antibodies used for SUnSET/FACS analyses: Anti-Puromycin (Millipore-MABE343). Secondary antibodies for IF: AF488 DONKEYF(AB')2, anti-mouse IgG (H+L) (Invitrogen).