![Fig. 6. Expression of the nonphosphorylatable mutant eIF-NP rescued PKR-mediated inhibition of protein synthesis in FANCC−/− cells. / (A) Analysis of phosphorylated eIF-2α levels in PKR-expressing MEFs carrying vector alone (VEC), wild-type eIF-2α (eIF), and a nonphosphorylatable mutant eIF-NP; cells were pretreated with 10 ng/mL of murine recombinant IFN-γ, transfected with 100 μg/mL of poly(I).poly(C), and labeled in a methionine-cysteine-free medium containing 50 μCi/mL of [35S]methionine-cysteine labeling mix for 60 minutes. Whole cell lysates were prepared, and 800 μg of total proteins from each sample were subjected to immunoprecipitation with anti–eIF-2α antibody. The immunocomplexes were analyzed by SDS-PAGE followed by autoradiography. NS, nonspecific. (B) Immunoblot analysis of a fraction of the same immunoprecipitates described in (A) using anti–eIF-2α antibody. HC, IgG heavy chain. (C) Rate of protein synthesis in PKR-expressing MEFs carrying vector alone (VEC), wild-type eIF-2α (eIF), and a nonphosphorylatable mutant eIF-NP. Cells were pretreated with 10 ng/mL of murine recombinant IFN-γ, transfected with 100 μg/mL of poly(I).poly(C), and labeled in a methionine-cysteine-free medium containing 50 μCi/mL of [35S]methionine-cysteine labeling mix for 60 minutes. Protein synthesis was measured by the incorporation of [35S]methionine and [35S]cysteine into trichloroacetic acid-precipitable proteins. The control cells did not carry any expression vectors. ▧, FANCC−/−; ■, FANCC+/+. Data represent means ± standard deviations of 2 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/97/6/10.1182_blood.v97.6.1644/6/h80610772006.jpeg?Expires=1735857294&Signature=bMS-V4gSM9kQQGdBfNb80P~tWUVtvPnfXYIXOt~gK1OZre8qDGKBh05OLNExcPqkPrsccIynxZOrZJJccjDggoI~jjV1Z5xguSg~8t1N9LJWp5p-TTd5uaCjcdnjvU7xqSrqjKOxctxL-76on6rsMT6JomXsWiW4JFLYP2tDmqpfs6yW-bvpGm61fSs4~pcBcJ9lDtGu33OvRiaRn1HGfnGF5H3HOQ1GbTOdJaKDslvlmAgv2jHvO3Iy8sojBk4ijR~mza0EI6jcvBtc1rQtWqR8ee~qkF30VZZmyOcgKGU7kSIu-wP11kFocENlNNwqB~VZ31s1xM9eGzIYLrg8~w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Expression of the nonphosphorylatable mutant eIF-NP rescued PKR-mediated inhibition of protein synthesis in FANCC−/− cells.
(A) Analysis of phosphorylated eIF-2α levels in PKR-expressing MEFs carrying vector alone (VEC), wild-type eIF-2α (eIF), and a nonphosphorylatable mutant eIF-NP; cells were pretreated with 10 ng/mL of murine recombinant IFN-γ, transfected with 100 μg/mL of poly(I).poly(C), and labeled in a methionine-cysteine-free medium containing 50 μCi/mL of [35S]methionine-cysteine labeling mix for 60 minutes. Whole cell lysates were prepared, and 800 μg of total proteins from each sample were subjected to immunoprecipitation with anti–eIF-2α antibody. The immunocomplexes were analyzed by SDS-PAGE followed by autoradiography. NS, nonspecific. (B) Immunoblot analysis of a fraction of the same immunoprecipitates described in (A) using anti–eIF-2α antibody. HC, IgG heavy chain. (C) Rate of protein synthesis in PKR-expressing MEFs carrying vector alone (VEC), wild-type eIF-2α (eIF), and a nonphosphorylatable mutant eIF-NP. Cells were pretreated with 10 ng/mL of murine recombinant IFN-γ, transfected with 100 μg/mL of poly(I).poly(C), and labeled in a methionine-cysteine-free medium containing 50 μCi/mL of [35S]methionine-cysteine labeling mix for 60 minutes. Protein synthesis was measured by the incorporation of [35S]methionine and [35S]cysteine into trichloroacetic acid-precipitable proteins. The control cells did not carry any expression vectors. ▧, FANCC−/−; ■, FANCC+/+. Data represent means ± standard deviations of 2 independent experiments.
