PKR kinase activity assay

PKR activity assays were performed using an anti-PKR monoclonal antibody (71/10, R&D systems). HeLa cells were maintained in DMEM containing 10% fetal bovine serum. The cells were harvested when they were at 70% confluence. Cells were washed in ice-cold PBS and collected by centrifugation at 600 g for 5 min. Cell extracts were prepared in lysis buffer [20 mM Tris–HCl (pH 7.5), 5 mM MgCl₂, 50 mM KCl, 400 mM NaCl, 2 mM dithiothreitol (DTT), 1% Triton-X 100, 100 U/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 20% glycerol]. A 100 µg aliquot of total protein was immunoprecipitated using the anti-PKR monoclonal antibody (71/10) in high-salt buffer [20 mM Tris–HCl (pH 7.5), 50 mM KCl, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% Triton-X 100, 100 U/ml aprotinin, 0.2 mM PMSF and 20% glycerol] at 4°C for 30 min on a rotating wheel. A 20 µl aliquot of Protein-A agarose beads was then added and incubated for 1 h. The Protein-A agarose beads were washed four times in 500 µl of high-salt buffer and twice in activity buffer [20 mM Tris–HCl (pH 7.5), 50 mM KCl, 2 mM MgCl₂, 2 mM MnCl₂, 0.1 mM PMSF and 5% glycerol]. The PKR assay was performed with PKR still attached to the beads in activity buffer containing 0.1 mM ATP and 1 µCi of [γ³²P] ATP at 30°C for 10 min. PKR was activated using synthesized TAR RNA (IDT DNA Technologies), and the effect of PACT, Tat, ADAR1 and TRBP on TAR-activated PKR was assayed by the subsequent addition of increasing amounts of pure recombinant PACT or pure recombinant TRBP (4, 40, 400 pg and 4 ng) in the presence of recombinant Tat and increasing amounts of recombinant ADAR1 (1.5, 15 and 150 ng). Labeled proteins were analyzed by SDS–PAGE on a 12% gel followed by autoradiography.