Figure 4 : MANF binds to the DNA binding domain of p65 through its C-terminal SAP-like domain.

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

Figure 4

(a) Schematic illustration of p65 and its truncates. RHD, Rel homology domain; NLS, nuclear localization signal; TAD, transactivation domain. (b) Mapping the domain of p65 binding to MANF by semi-pull-down assay. MANF-GST bound to glutathione beads was used as an affinity matrix to absorb full-length GFP-tagged p65 and its truncates expressing in 293T cells. The proteins were blotted with an anti-GFP antibody. (c) Confirmation of the binding site of p65 and MANF by in vitro pull-down assay. MANF-GST bound to glutathione beads were used as an affinity matrix for absorbing 6 × His-tagged p65-N2 fragment. The SDS-PAGE gel was stained with Coomassie brilliant blue (upper panel) and then blotted with an anti-His antibody (lower panel). (d) Schematic illustration of MANF and its truncates. (e) Mapping the binding site of MANF and p65 by co-immunoprecipitation. 293T cells were co-transfected as indicated, and treated with tunicamycin for 12 hrs at 24 hrs after transfection. (f) Confirming the binding site of MANF and p65 by pull-down assay. MANF-GST bound to glutathione beads were used as an affinity matrix for absorbing His-tagged p65-N2 fragment. The SDS-PAGE gel was stained with Coomassie brilliant blue (upper panel) and then blotted with anti-His antibody (lower panel). The experiments in panel b-c and e-f were repeated for at least three times.