Phosphor eif2α ser51

Approximately ten A₆₀₀ cells (∼10 ml of cells at A₆₀₀ ∼1) were collected from the respective cultures, pelleted, and flash-frozen in liquid nitrogen until further use. The cells were resuspended in 400 μl of 10% TCA and lysed by bead beating three times: 30 s of beating and then 1 min of cooling on ice. The precipitates were collected by centrifugation, resuspended in 400 μl of SDS-glycerol buffer (7.3% SDS, 29.1% glycerol and 83.3 mm Tris base), and heated at 100 °C for 10 min. The supernatant after centrifugation was treated as the crude total protein extract. Protein concentrations from extracts were estimated using a bicinchoninic acid assay (Thermo Scientific). Equal protein amounts of each sample was resolved on 4–12% Bis-Tris gels (Invitrogen). Coomassie Blue–stained gels were used as loading controls. Western blots were developed using antibodies against the respective tags. The following primary antibodies were used: monoclonal FLAG M2 (F3165, Sigma), HA (12CA5, Roche), and phosphor-eIF2α Ser51 (9721, Cell Signaling). Horseradish peroxidase–conjugated secondary antibodies (mouse and rabbit) were obtained from Sigma. The molecular weight markers used were 2661 (Thermo Scientific) and PG-P MT2922 (Genetix). For Western blotting, standard enhanced chemiluminescence reagents (GE Healthcare) were used. ImageJ was used for quantification.

Protein levels were quantified using an automated capillary electrophoresis and immunoassay system, Wes (ProteinSimple, CA, USA), as described previously 12. We used the following primary antibodies: PRDM14 (ab187881) and 78‐kDa glucose‐regulated protein (GRP78; ab21685) from Abcam Inc., C/EBP homologous protein (CHOP; #2895), GAPDH (#5174), eukaryotic initiation factor 2 α‐subunit (eIF2α; #5324), and phosphor‐eIF2α (Ser51, #3398) from Cell Signaling Technology Inc. The specificity of the PRDM14 antibody was confirmed previously by western blotting using knockdown cells 12.