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On resolving the molecular identity of the endothelial cell nucleosome assembly protein
Author(s) -
Clark Jennifer e,
Alexeyev Mikhail,
Stevens Troy
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1433-d
Subject(s) - nap , gene knockdown , microbiology and biotechnology , chemistry , small hairpin rna , endothelial stem cell , biology , gene , biochemistry , neuroscience , in vitro
Nucleosome assembly protein (NAP) is a chromatin‐associated protein that fulfills an important role in cell memory, and has been incriminated in the control of cell growth. We have recently shown that NAP expression is an important determinant of the endothelial vasculogenic phenotype. Western analysis using a monoclonal antibody specifically targeting the 4a8 epitope of NAP has consistently resolved two bands. The higher molecular weight band is 58 kD, and corresponds to full length NAP. The lower molecular weight band is 53 kD, and has been postulated to be a degradation product, although this lower band has yet to be specifically identified. To resolve the molecular identity of both NAP bands, full‐length NAP with a hemaglutinin tag was expressed in pulmonary artery endothelial cells (PAEC). Western analysis of PAEC nuclear and whole cells lysates using a hemaglutinin antibody resolved only a 58 kD band, indicating the 53 kD product is not strictly a degradation product. Short hairpin RNA (shRNA) was then utilized to knockdown NAP expression in pulmonary microvascular endothelial cells. This knockdown strategy resulted in disappearance of the 53 kD band in nuclear extracts, and decreased the 58 kD band. These data suggest that full‐length NAP encodes for the 58 kD product, and while the 53 kD product is related to NAP, it may be derived through either differential processing of the full‐length mRNA product, or result from expression of a related gene. Supported by:HL66299.