Fig 1: Western blot results using samples generated via the Rapid Protein Extraction (RPE) Method. A) MEFs were grown in cell culture flasks to ≤80% confluence in high glucose DMEM medium (Gibco Thermo Fisher Scientific, # 11965-092), supplemented with sodium pyruvate (1:100, 100 mM, Gibco Thermo Fisher Scientific, # 11360-070), penicillin and streptomycin antibiotic mix (1:100, 10,000 U/Ml, PenStrep, Gibco Thermo Fisher Scientific, # 15140-122), and fetal bovine serum (FBS) (1:10, heat inactivated at 60 °C for 1 h, Gibco Thermo Fisher Scientific, # 10270106). The medium was removed, the cells washed with PBS, detached from the flasks via a 5 min treatment with 0.25% trypsin (Gibco Thermo Fisher Scientific, # 15050-057), and then counted in a hemocytometer. 5 × 105 MEFs were seeded into 6 cm diameter plastic dishes in 4 ml of the above medium. The next day, the medium was removed, the dish washed with 4 ml PBS, followed by the addition of 4 ml DMEM medium lacking Leu (Vitrocell Embriolife, Brazil, # 00367, lacking Gln, Pyr and Leu), supplemented with Pyruvate, Gln (1:100, 200 mM, Gibco Thermo Fisher Scientific, # 25030-081), and dialysed FBS (1:10, heat inactivated as above, Gibco Thermo Fisher Scientific, # 26400-044) (three independent samples labelled A, B, C), or the same medium but containing Leu (Vitrocell Embriolife, Brazil, # 02209) (sample labelled as 0), or DMEM medium containing 2 μM MG132 (Sigma Aldrich, #M7449, diluted in DMSO) (three independent samples, labelled A, B, C). After the indicated incubation time, cells were subjected to the RPE Method. 20 μl of each sample was subjected to SDS-PAGE using a 6% denaturing acrylamide gel, and then the proteins transferred to a nitrocellulose membrane (Hybond-C Extra 0.45 μm, Amersham GE Healthcare Life Sciences, # RPN303E, or 0.2 μm Nitrocellulose membrane, Bio-Rad, # 1620112) via the wet transfer method (Mini Trans-Blot Electrophoretic Transfer Cell, Bio-Rad), at 100 V for 1 h in Tris Glycine buffer (25 mM Tris base, 192 mM glycine) containing 0.1% SDS. The membrane was subjected to immunoblotting, as described in Fig. 3, using antibodies against the phosphorylated form of Gcn2 (GCN2-P. 1:1000, Abcam, # ab75836). Thereafter, the membrane was treated with 15% H2O2 (30% H2O2 diluted in H2O, Ajax Finechem, Thermo Fisher Scientific, # 260) for 30 min (modified from [8]), extensively washed, and probed with antibodies against GCN2 [9]. B) Gcn2+/+ and Gcn2−/− MEFs were grown and samples generated as above, subjected to SDS-PAGE using a 6% gel, and to immunoblotting using antibodies against Gcn2, Gcn2-P, actin (1:5000, Sigma-Aldrich, # A2066), and Methionyl-tRNA Synthetase (MRS, 1:2000, Abcam, # ab50793). C) Samples were generated as above, and 5 μl of each sample subjected to SDS-PAGE using a 10% gel, and to immunoblotting using antibodies against eIF2α (1:1000, Thermo Fisher Scientific, # AHO0802), the phosphorylated form of eIF2α (eIF2α-P, 1:2000, Thermo Fisher Scientific, # 44-728G). eIF2α-P was detected first, the membrane treated with H2O2 as outlined in A) and then probed for eIF2α.