Overcoming off-targets: assessing Western blot signals for Bcnt/Cfdp1, a tentative component of the chromatin remodeling complex

• Published in: Bioscience reports Vol. 40; no. 6
• Main Authors: Iwashita, Shintaro, Suzuki, Takehiro, Kiriyama, Yoshimitsu, Dohmae, Naoshi, Ohoka, Yoshiharu, Song, Si-Young, Nakashima, Kentaro
• Format: Journal Article
• Language: English
• Published: England Portland Press Ltd 26.06.2020
• Subjects: Animals; Antibodies - immunology; Antibody Specificity; Biochemical Techniques & Resources; Blotting, Western; Brain - metabolism; Cattle; Chemical Biology; Chromatin Assembly and Disassembly; Epitopes; Female; Gene Expression Regulation, Developmental; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Molecular Bases of Health & Disease; Molecular Weight; Mouse Embryonic Stem Cells - metabolism; Nuclear Proteins - genetics; Nuclear Proteins - immunology; Nuclear Proteins - metabolism; Phosphorylation; Placenta - metabolism; Post-Translational Modifications; Pregnancy; Rats, Wistar; Structural Biology; Mass spectroscopy; Phosphoprotein; Western blot analysis; antibody evaluation; endogenous Bcnt/Cfdp1; RNA sequence analysis
• ISSN: 0144-8463; 1573-4935
• DOI: 10.1042/bsr20194012

The Bucentaur (BCNT) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in yeast and Drosophila. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the SNF2-related CBP activator protein (Srcap) chromatin remodeling complex, but little is known about its properties, partly because few antibodies are available to examine the endogenous protein. In this paper, we assigned the Western blot signal against the mouse Bcnt/Cfdp1 as a doublet of approximately 45 kDa using anti-Bcnt/Cfdp1 antibodies, which were generated against either of two unrelated immunogens, BCNT-C domain or mouse N-terminal peptide, and in addition, the Cfdp1 knockdown mouse ES cell line and bovine tissue were used as potential negative controls. Moreover, LC-MS/MS analysis of the corresponding doublet to the Flag-tagged mouse Bcnt/Cfdp1 that was constitutively expressed in a HEK293 cell exhibited that the upper band was much more phosphorylated than the lower band with preferential Ser phosphorylation in the WESF motif of BCNT-C domain. Western blot analysis with these evaluated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development of mouse and rat, which is consistent with a data file of the expression of Bcnt/Cfdp1 mRNA.