Immunoblot Analysis of PKR Protein

Immunoblot analysis was carried out to evaluate the accumulation of PKR protein. The procedure was published previously [31 (link)]. Briefly, total cells lysates were prepared from cells by SDS sample buffer 1X [62.5 mM Tris-HCl (Tris(hydroxymethyl)aminomethane hydrochloride) pH 6.8; 50 mM DTT (dithio-threitol); 10% glycerol; 2% SDS (sodium dodecyl sulfate); 0.01% Bromophenol Blue; EDTA-free Protease Inhibitor Cocktail 1X (Roche, Basel, Switzerland)], sonicated, and boiled for 5 min. An equal amount of protein extracts was subjected to SDS-gel electrophoresis (SDS-PAGE), transferred to membranes (Bio-Rad Life Science Research, Hercules, CA, USA), and subjected to immunoblot analysis. Specific bands were visualized using Immobilon Classico Western HRP substrate (Merk, Millipore, Rahway, NJ, USA). Anti-GAPDH (sc-32233) and PKR (A12) (sc393038) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and secondary HRP-conjugated goat anti-rabbit IgG was purchased from Millipore.