Pg p mt2922

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.

Media was removed, and cells were briefly washed with sterile 1X PBS. Cells were incubated in RIPA buffer (#R0278; Sigma Aldrich) containing protease inhibitor (#P8340; Sigma Aldrich) for lysis. After 1 h in ice, cells were scraped using a plastic scraper, followed by repeated cycles of sonication (15 kW, two cycles of 15 s each). Cell debris and whole cell lysate were separated by centrifugation (10,000 g for 10 min). Protein concentration was estimated with Bradford assay (Absorbance at 595 nm). Protein samples were processed for SDS polyacrylamide gel electrophoresis with 10–250 KDa prestained protein ladder (#PG-PMT2922; Genetix) as a molecular weight reference. Proteins were transferred onto a nitrocellulose membrane at 15 V, 2.5 A for 30 min. Membrane was blocked with 5% bovine serum albumin (BSA) for 1 h. Membrane was probed overnight at 4°C by incubation with different monoclonal human/rat specific primary antibodies as specified in Supplementary Table S1.