FIG. 4.

eIF2α kinase PEK is required for translocation of NF-κB into the nucleus during ER stress. (A and B) PEK^+/+ MEF cells grown on glass slides were treated with thapsigargin for 6 h (Tg; panel A) or no stress (UT; panel B). Cells were prepared and the p65 subunit of NF-κB was visualized using rabbit polyclonal antibodies specific for this transcription factor, followed by goat anti-rabbit IgG conjugated with Rhodamine Red. NF-κB linked with Rhodamine Red was visualized by laser confocal microscopy. Cell nuclei were stained with DAPI mountain medium, and electronic images from the Rhodamine Red and DAPI were merged in the right figures. (C through F) PEK^−/− cells transfected with a plasmid expressing PEK (PEK) in combination with plasmid encoding GFP were grown on glass slides. Transfected cells were exposed to thapsigargin (D and E) or were untreated (C). NF-κB was visualized by using the secondary antibody conjugated with Rhodamine Red, nuclear DNA by DAPI stain, and GFP by fluorescein isothiocyanate. All three panels were merged as indicated. PEK^−/− cells transiently expressing PEK were identified by coexpression with GFP. The PEK^−/− MEF cell in panel D was expressing no PEK or GFP and served as a control for eIF2α kinase dependence for NF-κB translocation to the nucleus during ER stress.