Fig. 5. Nuclear export of C/EBPβ-PSer-239 is mediated by CRM1 in hepatocytes treated with TNFα. Day 5 primary hepatocytes were isolated form C/EBPβ–/– mice. Representative confocal microscopy (n = 5 in each group). (A) Mouse hepatocytes were transfected with C/EBPβ, treated as described in Materials and methods and stained with antibodies against HA and CRM1. In C/EBPβ and TNF-α + leptomycin B cells, C/EBPβ (red) is nuclear and CRM1 (green) is nuclear and perinuclear. In TNF-α cells, C/EBPβ and CRM1 are co-localized in the cytoplasm (in yellow). Nuclei are stained with TOTO-3 (blue). (B) Mouse hepatocytes were transfected with CMV vectors expressing C/EBPβ-Ala239 or C/EBPβ-Asp239 for 24 h, treated as described in Materials and methods and stained with antibodies against HA and CRM1. C/EBPβ-Ala239 localized to the nucleus (in red) in untreated and TNF-α-treated hepatocytes. C/EBPβ-Asp239 co-localized with CRM1 in the perinuclear region (in yellow). Nuclei are stained with TOTO-3 (blue). (C) Cells were stained with antibodies against C/EBPα and CRM1. Control hepatocytes expressed nuclear C/EBPα. TNF-α treatment induced the nuclear export of C/EBPα, which co-localized with CRM1 in the perinuclear region (in yellow). Nuclei are stained with TOTO-3 (blue). (D) Mouse hepatocytes were treated with TNF-α or SIN-1 as described in Materials and methods, and stained with antibodies against HA and PSer239. TNFα and SIN-1 treatments induced the phosphorylation of C/EBPβ on Ser239 and its nuclear export. The cytoplasmic co-localization of C/EBPβ-PSer239 (green) and C/EBPβ (red) antibodies is shown in yellow. Control hepatocytes displayed mainly nuclear C/EBPβ (red) but not C/EBPβ-PSer239. Nuclei are stained with TOTO-3 (blue).