Figure 4. Phosphorylation of eIF2α facilitates lowered levels of IκBα in response to UV irradiation.

(A) GCN2^+/+ and GCN2^−/− MEF cells were exposed to 40 J/m² UV-C irradiation or no stress (0), and incubated in the culture medium for up to 8 h as indicated. Alternatively, the MEF cells were incubated with TNFα for 30 min. Equal amounts of proteins were analysed in each lane. Levels of phosphorylated IκBα, total IκBα, p65 and actin were measured by immunoblot analysis using polyclonal antibody specific to each protein. (B) Levels of protein synthesis were measured in GCN2^+/+ and GCN2^−/− MEF cells subjected to 40 J/m² UV-C irradiation or no stress (0). Following the UV stress, cells were incubated in the culture medium for the indicated time. Thirty minutes prior to harvesting, cells were radiolabelled with [³⁵S]Met/Cys. Cells were then washed, and radiolabelled proteins were separated by SDS/PAGE, and visualized by autoradiography. IκBα and p65 were immunoprecipitated using protein-specific antibodies, subjected to SDS/PAGE, and imaged by autoradiography. To show that equal amounts of proteins were analysed in the electrophoretic analysis, actin was measured by immunoblot analysis using the radioactive lysate preparation. Levels of general protein synthesis, IκBα and p65 in the GCN2^+/+ and GCN2^−/− MEF cells are listed relative to 100% synthesis in the non-stressed cells. (C) The turnover of IκBα was measured by radiolabelling GCN2^+/+ or GCN2^−/− MEF cells with [³⁵S]methionine/[³⁵S]cysteine for 30 min. Radiolabelled MEF cells were washed, incubated in DMEM supplemented with non-radiolabelled amino acids, and then exposed to 40 J/m² UV-C or no stress (0 min). Following incubation for between 20 and 160 min, IκBα was immunoprecipitated, separated by SDS/PAGE and visualized by autoradiography. Total levels of actin were measured by immunoblot to insure that equal amounts of lysates were analysed in each experimental lane. Results shown in (A)–(C) are representative of three experiments.