Transiently overexpressed Zfra sequesters NF-κB, WOX1, p53 and ERK in the cytoplasm. Breast MCF7 and neuroblastoma SK-N-SH cells were electroporated with an EGFP-Zfra construct or EGFP only, cultured overnight, and exposed to TNF for 40 min. Expression of GFP and GFP-Zfra is shown (A, B). (A) Without stimulation, EGFP-Zfra alone inhibited nuclear localization of NF-κB (p65), WOX1, p53 and p-ERK (Y204 phosphorylated) in SK-N-SH cells, whereas it had little or no effect on JNK1/2. TNF (50 ng/ml) could not induce nuclear translocation of endogenous JNK1/2, NF-κB (p65), p-ERK, p53 and WOX1 in Zfra-expressing cells during treatment for 40 min. (B) Similarly, in MCF7 cells, EGFP-Zfra suppressed nuclear localization of NF-κB and WOX1, and TNF could not induce nuclear translocation of these proteins. (C) EGFP-Zfra did not have a significant effect on the nuclear localization of WOX1 in Molt4 T cells; however, it blocked UV light-induced nuclear translocation of WOX1. Ectopic expression of EGFP and EGFP-Zfra in the above cells is shown in each panel. The levels of cytosolic α-tubulin are regarded as loading controls.