Anti-ADA3 Antibody, clone 5C9/C8

Transcriptional adapter 3 (UniProt O75528; also known as ADA3 homolog, ADA3-like protein, Alteration/deficiency in activation 3, hADA3, STAF54, Transcriptional adapter 3-like) is encoded by the TADA3 (also known as ADA3, TADA3L) gene (Gene ID 10474) in human. Alteration/deficiency in activation 3 (ADA3) is a component of several transcriptional co-activator and histone acetyltransferase (HAT) complexes and plays a critical role in in cell cycle regulation. Ada3 deletion in mice is embryonic lethal, and Ada3-deficient mouse embryonic fibroblasts (MEFs) exhiit a severe proliferation defect, dramatic changes in global histone acetylation, mitotic defects, as well as a delay in G2/M-to-G1 and G1-to-S transition. ADA3 also plays a role in genomic stability by controlling DNA repair checkpoints. ChIP-seq analsis reveals that ADA3 is significantly associated with human centromere regions across most chromosomes. In addition, ADA3 is found associated with CENP-B throughout all phases of the cell cycle, and CENP-B centromere binding decreased upon ADA3 knockdown. Wild-type human ADA3, but not CENP-B-binding deficient ADA3 mutant, prevented cell proliferation defects in MEFs following endogenous mouse ADA3 knockown. ADA3 overexpression and mislocalization correlates with poor prognosis in breast cancer patients.

~55 kDa observed. 48.90 kDa (human and mouse isoform 1) calculated. The larger-than-calculated band size is consistent with that reported in the literature (Mohibi, S., et al. (2015). J. Biol. Chem. 290(47):28299-28310). Uncharacterized bands may be observed in some lysate(s).

Full-length recombinant human ADA3.

Anti-ADA3, clone 5C9/C8, Cat. No. MABE1057, is a highly specific mouse monoclonal antibody that targets TADA3 and has been tested in Chromatin Immunoprecipitation (ChIP), Immunocytochemistry, Immunohistochemistry (Paraffin), and Western Blotting.

Immunohistochemistry Analysis: A representative lot detected nuclear ADA3 immunoreactivity in formalin-fixed, paraffin-embedded human breast carcinoma tissue section (Courtesy of Vimla Band, Ph.D., University of Nebraska USA).

Chromatin Immunoprecipitation (ChIP) Analysis: A representative lot detected ADA3 occupancy at the X chromosome centromere HOR region, the kinetochore assembly site also occupied by CENP-A and CENP-B, using TERT-immortalized human mammary epithelial cell 76N-TERT chromatin preparation. shRNA-mediated ADA3 knockdown led to a reduction in CENP-B, but not CENP-A, recruitment to the HOR region (Mohibi, S., et al. (2015). J. Biol. Chem. 290(47):28299-28310).

Immunocytochemistry Analysis: A representative lot detected ADA3 nuclear immunoreactivity in untransfected, but not ADA3 shRNA-transfected, TERT-immortalized human mammary epithelial 76N-TERT cells by fluorescent immunocytochemistry staining of 4% formaldehyde-fixed cells. Dual fluorescent staining revealed ADA3 and CENP-B nuclear colocalization (Mohibi, S., et al. (2015). J. Biol. Chem. 290(47):28299-28310).

Western Blotting Analysis: Representative lots detected both the endogenous mouse ADA3 (mADA3) and the exogenously expressed human ADA3 (hADA3) in Ada3FL/FL MEFs virally infected to express FLAG-tagged full-length or a.a. 111-432 hADA3 constructs. Cre recombinase expression downregulated only mADA3, but not hADA3 (Mohibi, S., et al. (2015). J. Biol. Chem. 290(47):28299-28310; Mohibi, S., et al. (2012). J. Biol. Chem. 287(35):29442-29456).

Western Blotting Analysis: A representative lot detected ADA3 expression levels among a panel of mouse tissues (Mohibi, S., et al. (2012). J. Biol. Chem. 287(35):29442-29456).

Research Category
Epigenetics & Nuclear Function

Clone 5C9/C8 specifically detected Cre recombinase expression-induced ADA3 downregulation in Ada3FL/FL MEFs. Clone 5C9/C8 immunostained the nuclei of untransfected, but not ADA3 shRNA-transfected, 76N-TERT human mammary epithelial cells (Mohibi, S., et al. (2015). J. Biol. Chem. 290(47):28299-28310; Mohibi, S., et al. (2012). J. Biol. Chem. 287(35):29442-29456).

Format: Purified

Protein G purified.

Purified mouse IgG1 in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide

Stable for 1 year at 2-8°C from date of receipt.

Evaluated by Western Blotting in MCF7 cell lysate.

Western Blotting Analysis: A 1:1,000 dilution of this antibody detected ADA3 in 10 µg of MCF-7 cell lysate.

Concentration: Please refer to lot specific datasheet.

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