|Detection of Human BMI‑1 by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line. Gels were loaded with 30 μg of whole cell lysate (WCL), 20 μg of cytoplasmic (Cyto), and 10 μg of nuclear extracts (Nuc). PVDF membrane was probed with 0.1 µg/mL Mouse Anti-Human BMI‑1 Monoclonal Antibody (Catalog # MAB33341) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band for BMI‑1 was detected at approximately 45 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 4.|
Chromatin Immunoprecipitation (ChIP)
|Detection of BMI‑1-regulated Genes by Chromatin Immunoprecipitation. HeLa human cervical epithelial carcinoma cell line was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. BMI‑1/DNA complexes were immunoprecipitated using 5 μg Mouse Anti-Human BMI‑1 Monoclonal Antibody (Catalog # MAB33341) or control antibody (Catalog # MAB003) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Mouse IgG Secondary Antibody (Catalog # BAF007). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The hoxc13 promoter was detected by standard PCR.|
Intracellular Staining by Flow Cytometry
|Detection of BMI‑1 in HeLa Human Cell Line by Flow Cytometry. HeLa human cervical epithelial carcinoma cell line was stained with Mouse Anti-Human BMI‑1 Monoclonal Antibody (Catalog # MAB33341, filled histogram) or isotype control antibody (Catalog # MAB003, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG F(ab')2 Secondary Antibody (Catalog # F0102B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
BMI-1 (B cell-specific Moloney-MLV integration site #1) is a 45 kDa protooncogene that is a class II member of the Polycomb group of genes. It participates in the formation of a large multimeric complex termed PRC1 that inhibits target gene transcription. Loss of BMI-1 function precludes stem cells from self-replicating. Human BMI-1 contains an N-terminal RING-finger domain (aa 17-56), an NLS (aa 81-95) and a C-terminal Pro/Ser-rich region (aa 251-326). Human BMI-1 shares 99%, 97%, 99% and 99% aa sequence identity with bovine, mouse, feline and canine BMI-1, respectively.
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