AIM: To study the effects of IκBα and its mutants (IκBαM, IκBα243N, IκBαM244C) on NF-κB, p53 and their downstream target genes. The relationship of NF-κB, p53, and IκBα was further discussed.METHODS: pECFP-IκBα, pECFP-IκBαM (amino acides 1-317, Ser32, 36A), pECFP-IκBα243N (amino acides 1-243), pECFP-IκBα244C (amino acides 244-317),pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-α-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection,location patts of NF-κB, p53 and IκBα (IκBαM,IκBα243N, IκBα244C) were observed by a laser scanning microscope (LSM510/ConfoCor2, Zeiss). RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids.Effects of IκBα and its mutants on the transprition level of NF-κB, NF-κB downstream target gene TNF-α, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments β-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader.RESULTS: Cells that were transfected with p53-DsRed revealed a predominant nuclear localization.YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IκBα, CFP-IκBαM, and CFP-IκBα243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IκBα244C revealed a predominant nuclear localization.The mRNA levels of p65, TNF-α, p53 and Bax in CFP-IκBα transfected cells did not change significantly,while in YFP-p65/CFP-IκBα co-transfected cells, IκBαdecreased the transcription of p65 downstream gene TNF-α (2.24 ± 0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08 ± 0.891) (P ＜ 0.05).Phosphorylation defective IκBα (IκBαM) decreased the transcription levels of all the four genes compared with the control (P ＜ 0.05). The N terminus of IκBα(IκBα243N) increased the transcription of NF-κB (1.84± 0.176) and TNF-α (1.51 ± 0.203) a little bit. However,the C terminus of IκBα (IκBα244C) increased the transcription of NF-κB, TNF-α, p53 and Bax significantly(8.29 ± 1.662, 14.16 ± 2.121, 10.2 ± 0.621, 3.72 ±0.346) (P ＜ 0.05). The CCK-8 experiment also showed that IκBα244C and p53 synergistically mediate apoptosis.CONCLUSIONS: IκBα and its mutants (IκBαM,IκBα243N, IκBαM244C) have different effects on NF-κB and p53 signaling pathways, according to their different structures. IκBαM bounds with NF-κB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IκBα244C may be co-factor in inducing apoptosis. The C terminal of IκBα enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.