PKR is constitutively activated in FANCC^−/− cells.

(A) Expression of PKR in MEFs in response to IFN-γ and dsRNA. Whole cell lysates were prepared 48 hours after incubation with IFN-γ (10 ng/mL) and 24 hours after transfection with 100 μg/mL of poly(I).poly(C), separated on a 7.5% SDS-PAGE, and immunoblotted with anti-mPKR antibody. (B) In vivo phosphorylation of PKR in MEFs treated with IFN-γ and dsRNA. FANCC^+/− and FANCC^−/− MEFs were labeled, 24 hours after incubation with IFN-γ (10 ng/mL) and 6 hours after transfection with 100 μg/mL of poly(I).poly(C), with [³²P]orthophosphate (150 μCi/mL) for an additional 3 hours. Cells were lysed, and equal amounts of whole cell lysates (500 μg of total proteins each sample) were immunoprecipitated with antibody specific for mPKR. [³²P]phosphate-incorporated PKR immunocomplexes were analyzed by SDS-PAGE, followed by autoradiography (top panel). To ensure additionally that equivalent amounts of the immunocomplexes were measured, immunoblot analysis of equal fractions of the PKR immunoprecipitates was performed by using the mPKR monoclonal antibody (bottom panel). P-mPKR, phosphorylated mouse PKR; HC, IgG heavy chain. (C) dsRNA-agarose affinity chromatography showing significantly more PKR from FANCC^−/− cell lysates binding to dsRNA. Equal amounts of whole cell lysates (500 μg of total proteins each sample) were applied to a poly(I).poly(C)-agarose column. The bound proteins were eluted and analyzed by SDS-PAGE and immunoblot, using anti-mPKR antibody.