Anti pkr sc 6282

All cells were either mock-infected or infected with the indicated viruses (MOI = 3.0). One day post-infection, the cells were lysed in 1% sodium dodecyl sulfate (SDS). Equivalent lysate volumes were separated on 10% SDS-polyacrylamide gels, then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk dissolved in TBST (20M Tris, 150mM NaCl, 0.1% Tween 20, pH 7.4) for 1 hour and probed with one of the following primary antibodies: anti-PKR (sc-6282; Santa Cruz Biotechnology, Inc.), anti-phospho-PKR (ab32036; Abcam), anti-eIF2α or anti-phospho-eIF2α (Ser51) antibody (9722 and 9721, respectively; Cell Signaling Technology), anti-TRS1 999^{27 (link)}, or anti-actin (A2066; Sigma). All primary antibodies were diluted in TBST containing 5% BSA and incubated overnight at 4°C with the membrane. Membranes were washed with TBST three times for 5 mins and then incubated for 1 hour at room temperature with either donkey anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (A16110 or 62–6520, respectively; Invitrogen) at 1:10,000 in TBST containing 5% (w/v) nonfat milk. Proteins were detected using the Amersham chemiluminescent detection system (GE Healthcare) according to the manufacturer’s recommendations. Membranes were imaged using the iBright Imaging System (Invitrogen).