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

Shintaro Iwashita,Yoshimitsu Kiriyama,Takehiro Suzuki,Yoshiharu Ohoka,Kentaro Nakashima,Si-Young Song,Naoshi Dohmae

doi:10.1042/bsr20194012

Shintaro Iwashita, Yoshimitsu Kiriyama + Show 5 more

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https://doi.org/10.1042/bsr20194012

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Abstract

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.

Highlights

The BCNT family members in yeast and Drosophila have been shown to play essential roles in gene expression and chromosomal maintenance [1]
Mammalian Bcnt/Cfdp1 is presumed to be a component of the SNF2-related CBP activator protein (Srcap) chromatin remodeling complex, which is based on the results using fractionation of cultured human cell extracts followed by mass spectrometry [2]
We showed that the difference between its calculated and apparent molecular mass is mainly due to the acidic stretch located in the N-terminal region and Ser250 phosphorylation in the BCNT-C domain [16]

Summary

Introduction

The BCNT family members in yeast and Drosophila have been shown to play essential roles in gene expression and chromosomal maintenance [1]. Mammalian Bcnt/Cfdp is presumed to be a component of the SNF2-related CBP activator protein (Srcap) chromatin remodeling complex, which is based on the results using fractionation of cultured human cell extracts followed by mass spectrometry [2]. This presumed frame is originally based on the analysis of Swc, a budding yeast ortholog of Bcnt/Cfdp, in Swr (yeast Srcap) chromatin complex [3–5]. Swc is not essential for survival, yet its deletion mutant swc cells caused lack in histone replacement activity for Swr, resulting in genetic instability, hypersensitivity to drugs, and transcriptional misregulation [9], [The Saccharomyces Genome Database https://www.yeastgenome.org/]

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