Puromycin labeling and immunoblot analysis

For puromycin labeling experiments, 3 x 10⁵ cells were plated in 60 mm dishes. Two days later, culture media was changed to fresh media. For monitoring basal translation, the cells were treated with 10 μg/ml puromycin for 10 min and harvested. For monitoring translation in stimulated cells, cells were treated with 250 nM thapsigargin for 24 hours and 10 μg/ml puromycin was added during the last 10 min before harvest. For monitoring translation in recovering cells, the stimulated cells were washed by 3 ml of culture media and incubated for 1 hour, followed by treatment of 10 μg/ml puromycin during the last 10 min before harvest. Cells were lysed in a lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 10% Glycerol, 2 mM PMSF, 10 μg/ml aprotinin, 4 μg/μl pepstatin and 4 μM leupeptin. After centrifugation at 21130 x g for 15 min, supernatants were mixed with a standard SDS-PAGE sample buffer. 40 μg of total protein was subjected to 12.5% SDS–polyacrylamide gels electrophoresis and transferred onto Immobilin-P PVDF membrane (EMD Millipore). Immunoblot detection was conducted using primary antibodies for puromycinylated protein [26], total eIF2α [27], and IRDye 800 conjugated secondary antisera (LI-COR) followed by scanning on a Odyssey near infrared imager (LI-COR). Scanned images were quantified using imageJ software.

For detection of eIF2Bδ, a rabbit anti-eIF2Bδ primary antibody (Proteintech), HRP-linked rabbit secondary antibody and SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher) were used.