Figure 6 : MANF represses the binding of p65 to its target genes.

From: Mesencephalic Astrocyte-Derived Neurotrophic Factor Is Involved in Inflammation by Negatively Regulating the NF-κB Pathway

Figure 6

(a) MANF does not influence the total level of IκBα induced by TNF-α. 293T cells were transfected with MANF-FLAG and control vector, then treated with TNF-α (10 ng/ml) for the indicated times. Western-blotting was performed to check the level of IκBα. (b) MANF does not affect TNF-α-induced nuclear translocation of p65. 293T cells were transfected with MANF-GFP or GFP vector (as a control), and treated with TNF-α (10 ng/ml) for 30 min. Cytoplasmic and nuclear fractions were prepared and immunoblotted with the indicated antibodies, respectively. (c) MANF represses the DNA-binding activity of p65. The cells were transfected with the indicated plasmids for 36 hrs and treated with TNF-α for 10-30 min. Nuclear NF-κB was examined by EMSA. Sp1 was used as a loading control. (d) Competition analysis used in EMSA. 100-fold excess of unlabeled wild-type or mutant κB probes were added to the reaction mixtures. (e) MANF represses the association of p65 with its target promoters. 293T cells were transfected with MANF-siRNA for 36 hrs and treated with TNF-α for 30 min. ChIP assay was performed by using the indicated antibodies. NC-si, NC-siRNA; MANF-si, MANF-siRNA. The experiments in all the panels were repeated for at least three times.