Journal: Communications Biology

Article Title: Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor

doi: 10.1038/s42003-021-02924-2

Figure Lengend Snippet: Shown are the general domain structures of Capsaspora NF-κB and the choanoflagellate RHD-only proteins as compared to mammalian NF-κBs. Green, RHD (Rel Homology Domain); Purple, nuclear localization sequence; Blue, GRR (glycine-rich region); Black bars, Ankyrin repeats; Pink, sequences in choanoflagellates that are not typically seen in other organisms.

Article Snippet: This was carried out by first preparing a transfection mix as follows: (1) 5 μg of pcDNA expression plasmid and 25 ul of 1 mg/ml PEI was added to 300 μl of PBS; (2) the mix was incubated for 15–20 min at room temperature with occasional swirling; (3) 2.2 ml of Capsaspora media (ATCC medium 1034) was then added to the mix; and media was removed from the 35-mm dish containing cells, and the transfection mix was added to the plate.

Techniques: Sequencing

Journal: Communications Biology

Article Title: Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor

doi: 10.1038/s42003-021-02924-2

Figure Lengend Snippet: Capsaspora cells were transfected with FLAG-tagged vectors for full-length Co -NF-κB, mutant Co -RHD, and mutant Co -Cterm. The cells were stained using anti-FLAG antiserum (left panels) and Hoechst (middle panels), and then merged (right panels). Scale bars are 2 µm. The dotted white lines indicate the periphery of the cells.

Article Snippet: This was carried out by first preparing a transfection mix as follows: (1) 5 μg of pcDNA expression plasmid and 25 ul of 1 mg/ml PEI was added to 300 μl of PBS; (2) the mix was incubated for 15–20 min at room temperature with occasional swirling; (3) 2.2 ml of Capsaspora media (ATCC medium 1034) was then added to the mix; and media was removed from the 35-mm dish containing cells, and the transfection mix was added to the plate.

Techniques: Transfection, Mutagenesis, Staining

Journal: Communications Biology

Article Title: Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor

doi: 10.1038/s42003-021-02924-2

Figure Lengend Snippet: a Protein binding microarray (PBM) DNA-binding profiles of Co -NF-κB as compared to Nematostella vectensis ( Nv ) NF-κB cysteine (cys) allele (top, left), human (Hu) RelA (top, right), human p52 (bottom, left), and human cRel (bottom, right). The axes are z-scores. Red dots represent random background sequences ( n = 1159), and blue dots represent NF-κB binding sites ( n = 2592). Black line is the best fit line (Co-NF-κB vs. the following: Nv-NF-κB, R 2 = 0.23; Hu-p52, R 2 = 0.27; Hu-cRel, R 2 = 0.08; Hu-RelA, R 2 = 0.01). b The consensus DNA-binding motif of Co -NF-κB generated from the PBM data in a . The motif was generated using the MEME motif discovery package using the 25 highest scoring binding sites identified by the PBM experiment in a . c Top: The FPKM values from Sebé-Pedrós et al. of NF-κB at each life stage, done in triplicate. Agg, Aggregative (yellow), Filo, Filopodic (green), Cys, Cystic (blue). Error bars are standard deviation. Bottom: Images taken with a light microscope of each life stage (Agg, Filo, and Cys from left to right). Yellow scale bar is 20 µm. Raw data are in Supplementary Data . d Capsaspora whole-cell extracts were created from each life stage (see Methods). 70 µg of each extract was then used in an electromobility shift assay, a palindromic κB-site probe (GGGAATTCCC). Lane 1 contains only free probe (-). Lanes 2-4 contain lysates from Agg, Filo, and Cys life stages incubated with a radioactive κB-site probe. Lanes 6–8, and lanes 10–12 contain lysates from Agg, Filo, and Cys life stages as indicated, and were incubated with an excess (10× and 25×, respectively) of unlabeled κB-site probe. Lane 13 contains the Cys lysate incubated with 25× unlabeled IRF-site probe. Lanes 5 and 9 contain no samples. NF-κB complexes and free probe are indicated with arrows. The dashed lines indicate where the image was cut to remove excess lanes. Raw image is in Supplementary Fig. . e The expression profiles of the indicated genes correlate with NF-κB mRNA expression in each life stage (Agg, low; Filo, medium; Cys, high), and were identified as developmental and immune system genes via Biological Processes GO analysis. Two of the genes in this list ( SRMS and SLK ) also contain κB sites in the 500 bp upstream of their TSS.

Article Snippet: This was carried out by first preparing a transfection mix as follows: (1) 5 μg of pcDNA expression plasmid and 25 ul of 1 mg/ml PEI was added to 300 μl of PBS; (2) the mix was incubated for 15–20 min at room temperature with occasional swirling; (3) 2.2 ml of Capsaspora media (ATCC medium 1034) was then added to the mix; and media was removed from the 35-mm dish containing cells, and the transfection mix was added to the plate.

Techniques: Protein Binding, Microarray, Binding Assay, Generated, Standard Deviation, Light Microscopy, Electro Mobility Shift Assay, Incubation, Expressing