Total eif2a

Livers were homogenized in RIPA buffer (Cell Signaling Technology), supplemented with complete mini EDTA-free protease inhibitor (Sigma Aldrich) and PhosSTOP phosphatase inhibitor (Sigma-Aldrich) using a Bead Mill Homogenizer (VWR). Protein concentrations were measured using a DC Protein Assay (Bio-Rad). Proteins were incubated at 60°C for 15 min in 4× Laemmli sample buffer (Bio-Rad). Liver lysate protein of 20 to 40 μg was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with TBS-T containing 5% nonfat dry milk for 1 h at room temperature and then incubated overnight at 4 °C in primary antibody. Membranes were washed in TBS-T, incubated in secondary antibody, washed with TBS-T, and visualized using SuperSignal Chemiluminescent Substrate (Thermo Scientific). Band intensity was measured using ImageJ software. Antibodies used include total eIF2a (Cell Signaling Technology #9722, 1:1000, in 5% BSA), phosphor-Ser51-eIF2a (Cell Signaling Technology #9721, 1:1000), vinculin (Cell Signaling Technology #4650, 1:1000), ApoB (Abcam ab31992, 1:1000), and FIT2 (a generous gift from David Silver’s laboratory (1 (link)), 1:1000)

Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer containing a cocktail of protease (Roche) and phosphatase inhibitors (Roche), separated by SDS-PAGE, and transferred to Immobilon-P membranes (Millipore). The following antibodies were used: tubulin (Millipore), cleaved caspase 3 (Asp 175) (Cell Signaling), cleaved PARP (Asp 214) (Cell Signaling), total PARP (Cell Signaling), total JNK (Cell Signaling), phospho-JNK (T183/Y185) (Cell Signaling), total p38 (Cell Signaling), phospho-p38 (T180/Y182) (Cell Signaling), total MKK4 (Cell Signaling), phospho-MKK4 (Ser257/Thr261) (Cell Signaling), GRP78 (BD Biosciences), total eif2a (Cell Signaling), and phospho-eif2a (Cell Signaling), XBP-1 s (Cell Signaling), CHOP (Cell Signaling), and Kir6.2 (Santa Cruz). Quantification of western blot analysis was done using ImageJ.