Figure 4 | Scientific Reports

Figure 4

From: N-Myc-Interacting Protein Negatively Regulates TNF-α-Induced NF-κB Transcriptional Activity by Sequestering NF-κB/p65 in the Cytoplasm

Figure 4

NMI interacts with members of NF-κB family protein. (A) The 293 T cells were transfected with the Myc-p65 and FLAG-NMI plasmids. At 24 h posttransfection, the cells were lysed, immunoprecipitated with anti-FLAG (left panel) or anti-Myc (right panel) Abs, and immunoblotted with anti-Myc or anti-FLAG Abs. An aliquot of each total cell lysate was analyzed by immunoblotting with the indicated Abs. (B) Endogenous NMI interacts with p65. HeLa cells were lysed, the cell lysates were then immunoprecipitated with anti-NMI Ab, and immunoblotted with anti-p65 Ab. (C) Two-step co-IP assay. The HEK293T cells were transfected with the HA-p65, FLAG-NMI and Myc- IκBα plasmids as indicated. Cells were lysed. The first immunoprecipitation was performed with an anti-FLAG antibody. The complex was eluted with the FLAG peptide, followed by the second step of immunoprecipitation with an anti-HA antibody. Protein samples from each step were subjected to western blot analysis. (D–H) NMI interacts with members of NF-κB family proteins. HeLa cells were transfected with the indicated plasmids, and the cells were lysed, immunoprecipitated with anti-Myc Ab, and immunoblotted with anti-HA Ab.

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