Analysis of the expression pattern of Ebp1, an ErbB-3-binding protein

Ebp1, a member of the PA2G4 family, was isolated as an ErbB-3-binding prote in in our laboratory using yeast two hybrid analysis. Although Ebp1 mRNA is ubiquitously expressed, little is known about either the expression of Ebp 1 protein in vivo or its translation initiation site. Western blotting anal ysis of a wide range of cell lines and primary tissue indicated that in the majority of cases Ebp1 is expressed as a single protein which migrates at 48 kDa in SDS-polyacrylamide gels. We show using epitope-tagged expression constructs that the second, not the first, in-frame ATG is used for the ini tiation of translation of the endogenous protein, encoding a protein predic ted to be 41.5 kDa. The molecular mass of endogenous Ebp1 protein derived f rom mouse liver and brain was determined by mass spectrometry and the data confirm that translation of endogenous Ebp1 in tissues is initiated from th e second in-frame ATG. (C) 2001 Academic Press.