|Spi-B in Mouse Splenocytes. Spi-B was detected in immersion fixed mouse splenocytes using Sheep Anti-Mouse Spi-B Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7204) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to plasma membranes and cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.|
Intracellular Staining by Flow Cytometry
|Detection of SPi-B in Mouse Splenocytes by Flow Cytometry. Mouse splenocytes were stained with Sheep Anti-Mouse Spi-B Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7204) followed by Allophycocyanin-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # F0127) and Rat Anti-Mouse B220/CD45R PE-conjugated Monoclonal Antibody (Catalog # FAB1217P). Quadrant markers were set based on control antibody staining (Catalog # 5‑001‑A). To facilitate intracellular staining, cells were fixed with paraformadehyde and permeabilized with saponin.|
Spi-B (Transcription factor Spi-B) is a 33-45 kDa member of the ets family of transcription factors. It is found in hematopoietic cells such as B cells and plasmacytoid dendritic cells (DC). In transitional B cells, Spi-B promotes their differentiation into follicular (naïve) B cells. In hematopoietic stem cells, Spi-B stimulates the generation of IFN-producing plasmacytoid DC at the expense of T, B and NK cell development. Mouse Spi-B is 267 amino acids (aa) in length. It contains a dual transactivation region (aa 1-62), plus a PEST domain (aa 110-170) and an Ets DNA-binding domain (aa 174-257). There are two isoform variants. One shows a nine aa substitution for aa 1-8, while a second possesses an 18 aa insertion after Leu17. Over aa 18-167, mouse Spi-B shares 91% and 74% aa identity with rat and human Spi-B, respectively.
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