Ab32052

PKR kinase activity was assessed using a combination of an EIF2AK2 kinase enzyme kit (Promega) and ADP‐Glo kinase assay (Promega). DCs were stimulated as previously described to induce kinase activity. After 2 h, whole‐cell extracts were prepared using PKR lysis buffer (50 mM Tris‐HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 0.5% Igepal, 12.5 mM beta‐glycerophosphate, 10% glycerol, 1 μg/mL leupeptin, 1 μg/mL pepstatin A, 0.4 mM PMSF). To capture PKR in whole‐cell extract, 96‐well high binding plates (Thermo Fisher) were coated with 10 μg/mL anti‐PKR (ab32052, Abcam) O/N at 4°C. Then, anti‐PKR‐coated wells were blocked with 1%BSA for 30 min and incubated with 10 μg whole‐cell extract for 2 h. Recombinant PKR was used to obtain a protein standard used for quantification (Promega). Wells were incubated with 0.1 mg/mL substrate (myelin basic protein) in 22.5 μL kinase activity buffer (40 mM Tris‐HCl [pH 7.5], 20 mM MgCl₂, 50 μM DTT, 0.1 mg/mL BSA, 50 μM ATP) for 1 h at RT. Kinase activity termination and ATP depletion were achieved using the ADP‐Glo reagent (Promega) for 40 min at RT. Final PKR kinase activity was measured by incubation with kinase detection reagent for 30 min at RT, after which the mixture was transferred to a white opaque plate (Corning). Relative light units were detected as a measure for generated ADP and, thus, PKR activity, using the BioTek Synergy HT.