Western blot analysis.

Cells were collected by scraping at the indicated times and frozen as dry pellets at −80°C until use. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, 1 mM EDTA) containing protease and phosphatase inhibitors (Roche), and the protein concentration was determined by the Bradford assay (Amresco). Equivalent amounts of protein were resolved on SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham). For monoclonal antibodies, membranes were blocked for 1 h at room temperature in 1% bovine serum albumin (BSA) in TBS-T (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) before incubation with primary antibody in TBS-T with 1% BSA for 1 h at room temperature or overnight at 4°C. For polyclonal antibodies, membranes were blocked in 1% BSA followed by incubation with primary antibody overnight at 4°C in 5% BSA in TBS-T. For Western blots using the phospho-eIF2α (Ser51) antibody, membranes were blocked in 5% BSA in TBS-T overnight at 4°C, followed by overnight incubation at 4°C with primary antibody diluted in TBS-T containing 5% BSA. Antibodies to the following proteins were used in this study: IE1 (52) (1:10,000), pUL44 (1:1,000; Virusys), pp28 (53) (1:5,000), pTRS1 (40) (1:100), pIRS1 (40) (1:100), P99 antibody against pTRS1 and pIRS1 (17) (1:10,000), tubulin (1:50,000; Sigma T6199), total PKR (1:1,000; Santa Cruz sc-707), phospho-PKR (Thr446) (1:1,000; Abcam ab32036), total eIF2α (1:1,000; catalog no. 3072; Cell Signaling), and phospho-eIF2α (Ser51) (1:1,000; catalog no. 3398; Cell Signaling).