Mouse anti ha 12ca5

Yeast strains were grown to mid-log phase in SD-His medium, treated, or not, with 20 mM 3-AT for 30 min, collected by filtration, and frozen in liquid nitrogen. Cells were disrupted in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.1% NP-40, 60 mM b-glycerophosphate, 1 3 PIC, and 1 3 Pefabloc) using a FastPrep-24TM, and the lysates were then clarified by centrifugation (3 0 220 3 g for 5 min at 4 C). Co-immunoprecipitation was performed with 10 mg of total protein using Pierce TM Anti-HA magnetic beads (Thermo Scientific). eIF2 subunits were detected using specific rabbit antisera (Perzlmaier et al., 2013) . For HA-tag and c-Myc-tag detection, mouse anti-HA 12CA5 or anti-c-Myc 9E10 (Santa Cruz Biotechnology) antibodies were used, respectively. Sui2-Ser 52 phosphorylation was monitored in whole cell lysates prepared as described (Hatakeyama and De Virgilio, 2019) using phospho-EIF2S1 (Ser 52 ) polyclonal antibody (Invitrogen). The list of primary and secondary antibodies with indicated working dilutions can be found in the Key resources table. ECL Western Blotting Detection (GE Healthcare) or Radiance Plus Sensitive ECL (Azure Biosystems) were used for the western blot development. The blots were quantified using ImageJ (NIH).