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. 2021 Dec 16;4:1404. doi: 10.1038/s42003-021-02924-2

Fig. 2. DNA-binding, subcellular localization, and transcriptional activation activities of Co-NF-κB.

Fig. 2

a FLAG-tagged expression vectors used in these experiments. From top to bottom, the drawings depict the naturally shortened Nv-NF-κB, the full-length Co-NF-κB protein, an N-terminal-only mutant containing the RHD and GRR (Co-RHD), and a C-terminal-only mutant containing the ANK repeats and other C-terminal sequences (Co-Cterm). b Anti-FLAG Western blot of lysates of HEK 293T cells transfected with the indicated expression vectors or the vector control (-). Raw image is shown in Supplementary Fig. 10. c Indirect immunofluorescence of DF-1 chicken fibroblasts transfected with the indicated expression vectors. Cells were then stained with anti-FLAG antiserum (left panels) and Hoechst (middle panels), and then merged in the right panels. Yellow scale bar is 10 µm. d A κB-site electromobility shift assay (EMSA) using a palindromic κB-site probe (GGGAATTCCC) and each of the indicated lysates from b. The NF-κB complexes and free probe are indicated by arrows. Raw image is shown in Supplementary Fig. 10. e A κB-site luciferase reporter gene assay was performed with the indicated proteins in HEK 293 cells. Luciferase activity is relative (Rel.) to that seen with the empty vector control (-) (set at 1.0). Values are averages of n = 3 biological replicates per sample, each of which was performed in triplicate, and are reported with standard error. Raw data are in Supplementary Data 5. f A GAL4-site LacZ reporter gene assay was performed in yeast Y190 cells with the indicated GAL4-fusion proteins or the GAL4 vector alone control (-). Values are the average β-gal units for n = 34 samples, except for 8 samples for Co-NF-κB, and are presented with standard errors. Raw data are in Supplementary Data 5. g Co-immunoprecipitation (IP) assays of MYC-tagged Co-Cterm. MYC-Co-Cterm was co-transfected in HEK 293T cells with pcDNA FLAG or FLAG-Co-RHD as indicated. An IP using anti-FLAG beads was next performed. After isolating proteins on FLAG beads and separating proteins on SDS-polyacrylamide gels, Western blotting with anti-FLAG (top) and anti-MYC (middle) antibodies was then performed. An anti-MYC Western blot of the whole-cell (WC) lysates was also performed (bottom). Raw image is shown in Supplementary Fig. 10. h Indirect immunofluorescence of DF-1 chicken fibroblasts transfected with the indicated expression vectors. Shown are four representative cells that were co-stained with anti-FLAG antiserum (left panel, Alexa fluor-488), anti-MYC antiserum (second panel, Alexa flour-555), Hoechst (third panel), and then merged (right panel).