293T transfectants, transfected with a siRNA mix, as described above, were harvested 48 h after the transfection, washed in PBS and lysed in 100 ml of Ripa-Doc (final concentration: 140 mM NaCl, 8 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.05% sodium dodecyl sulphate), containing a cocktail of protease and phosphatase inhibitors. Equal amounts of proteins (15 mg) were separated on a 10% SDS-PAGE gel, transferred on a nitrocellulose membrane and immunoblotted with a mouse anti-PKR hybridoma supernatant (clone F9) (a generous gift from A. Koromilas, McGill University) and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Amersham) diluted 1/1500. After detection of the antigen-antibody complexes, the membrane was washed with 25 ml of stripping buffer (final concentration: 0.08 M b-mercaptoethanol, 2% sodium dodecyl sulphate and 0.06 M Tris-HCl, pH 6.9) for 30 min at 508C, and immunoblotted with a mouse antia-tubulin monoclonal antibody (clone B-5-1-2 Sigma) diluted 1/5000 and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody diluted 1/1500. Antigen-antibody complexes were detected with an enhanced chemiluminescence (ECL) system.