Pkr a12 sc393038

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.

Polyclonal antibodies against the housekeeping gene GAPDH (sc-32233), GFP (B-2) (sc-9996) and PKR (A12) (sc393038) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Anti-PKR (phospho-T446, ab 32036) was purchased from Abcam (Cambridge, England). Antibodies against the viral proteins VHS and U_S3, were kindly provided by Professor Bernard Roizman. Secondary antibodies anti-rabbit and anti-mouse IgG conjugated to peroxidase were purchased from Merck Millipore. Polyinosinic-Polycytidylic acid [poly (I:C)] was used to induce activation of PKR and was provided by Sigma Aldrich (P1530). 293T cells were transfected as described above and 24 h post transfection, the cells were treated with 10 μg/ml of poly (I: C) diluted in OptiMEM. Samples were then collected at indicated time post transfection to perform immunoblot analysis. Actinomycin D (A9415), Cycloheximide (01810) and Phosphonoacetic acid (P6909) were purchased from Sigma-Aldrich.