Tissues or cultured cells were rinsed in ice-cold PBS and were immediately homogenized in 5 vol Triton X-100 buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM tetrasodium pyrophosphate, 100 mM NaF, 17.5 mM β-glycerophosphate, 10 mM PMSF, 15 μg/ml aprotonin, and 6 μg/ml pepstatin A) using a motorized homogenizer. After incubating on ice for 15 min, the extracts were cleared by centrifugation at 14,000 rpm twice for 30 min each. The protein content of each extract was determined by protein assay (Bio-Rad Laboratories). The extracts (40 μg) were separated by SDS-PAGE and were transferred to nitrocellulose. The blots were incubated with primary antibody (see below), and the signal was revealed by chemiluminescence after reacting with HRP-conjugated second antibody. The following primary antibodies were used: anti–eIF-2α (1:500; Santa Cruz Biotechnology, Inc.); anti–p-eIF-2α (1:1,000; Cell Signaling Technology); anti–caspase-12 (1:500; Santa Cruz Biotechnology, Inc.); and antiactin (1:1,000; Sigma-Aldrich).