Enforced expression of eIF-NP inhibits PKR-induced apoptosis in FANCC^−/− MEFs.

(A) Expression of HA-eIF proteins in FANCC^+/+ and FANCC^−/− cells. Whole cell lysates (80 μg of total proteins) were separated on a 7.5% SDS-PAGE and immunoblotted with the anti-HA antibody (top panel) or the anti–eIF-2α antibody (bottom panel). (B) Expression of HA-IκB proteins in FANCC^+/+ and FANCC^−/− cells. Total proteins (80 μg) were analyzed as described in (A), using the anti-HA antibody (top panel) or the anti–IκB-α antibody (bottom panel). (C) Cell viability of PKR-expressing MEFs carrying vector alone (VEC), wild-type eIF-2α (eIF), a nonphosphorylatable mutant eIF-NP, wild-type IκB-α (IκB), or a nonphosphorylatable mutant IκB-M; data points were determined by trypan blue exclusion assay as described in Figure 1 and presented as the mean (standard deviations of triplicate determinations. ▧, FANCC^−/−; ■, FANCC^+/+. (D) Expression of hPKR and eIF proteins in FANCC^+/+ and FANCC^−/− cells. Total proteins (80 μg) were analyzed by Western blotting with anti-PKR (top panel), anti-HA (middle panel), or anti–eIF-2α antibody (bottom panel). (E) Expression of hPKR and IκB proteins in FANCC^+/+ and FANCC^−/−cells. Total proteins (80 μg) were analyzed by immunoblotting with anti-PKR (top panel), anti-HA (middle panel), or anti–IκB-α antibody (bottom panel).