FIG 8.

PKR activation is independent of dsRNA binding during hyperosmotic stress. (A) Schematic representation of the WT and mutant PKR constructs (confirmed by sequencing of cDNA isolated from transfected cells), introduced stably into MEFs deficient in PKR. (B) Immunoblot analysis of FLAG-PKR expression in reconstituted MEFs. (C) Immunoblot analysis for the indicated proteins in cell extracts isolated from MEFs reconstituted with constructs shown in panel A and treated with hyperosmotic medium for the indicated durations. (D) Quantification of PKR-P(T451) bands normalized to total PKR bands from the experiment shown in panel C. (E) RT-qPCR analysis for the indicated mRNAs of RNA isolated from reconstituted MEFs and treated with hyperosmotic medium for the indicated durations. (F) Immunoblot analysis for the indicated proteins isolated from wild-type or mutant FLAG-PKR-reconstituted MEFs transfected with poly(I·C) for 6 h. (G) Immunoblot analysis for the indicated proteins isolated from either immunoprecipitations of FLAG-PKR from the indicated MEFs (IP) or from cell extracts before immunoprecipitations (input). All MEFs were treated with 600 mosM medium for 3 h. An antibody against PKR was used for the immunoblotting. (H) In vitro kinase activity assays on immunoprecipitated FLAG-PKR from cells treated as described for panel G. An equal volume of immunoprecipitated protein was used for the kinase assays. The kinase assay was performed in the presence or absence of the substrate GST-eIF2α, as indicated. FLAG-PKR autophosphorylation and the substrate GST-eIF2α^1–180 phosphorylation are shown at 60 kDa and 41 kDa, respectively. (I) In vitro kinase activity assays on immunoprecipitated FLAG-PKR from the indicated MEFs treated for 6 h with poly(I·C) (top) and autophosphorylated FLAG-PKR (bottom). Kinase assays were in the absence of substrate. Error bars represent standard errors of the means. *, P < 0.05; ns, not significant (Student's t test).