Loss of Immunostaining of the RBP-Jx Transcription Factor upon F Cell Differentiation Induced by Retinoic Acid1

Abstract

RBP-Jkappa is a novel type of transcriptional regulatory protein that does not contain any known DNA-binding motif. We raised anti-RBP-Jkappa monoclonal antibodies (K0043 and T6709) to investigate the roles of RBP-Jkappa in cell differentiation. These antibodies stained nuclei of undifferentiated embryonic stem cells and F9 cells but not those of the other differentiated cell lines tested so far although the RBP-Jkappa protein is expressed at similar levels. Interestingly, differentiated F9 cells lost the immunostaining reaction with the anti-RBP-Jkappa monoclonal antibodies. Biochemical subcellular fractionation study showed that the majority of RBP-Jkappa was localized in nuclei of F9 cells and that there are at least two forms of the RBP-Jkapppa protein in the nuclei of undifferentiated F9 cells, a free form and a chromatin-bound form. Upon induction of F9 cell differentiation, free nuclear RBP-Jkappa disappeared concomitantly with the loss of immunostaining, suggesting that the anti-RBP-Jkapppa antibodies cannot recognize chromatin-bound RBP-Jkappa. Since there is no evidence to indicate covalent modification of RBP-Jkappa, we assume that chromatin-bound RBP-Jkappa interacts with a large number of proteins which block the exposure of RBP-Jkappa epitopes to the monoclonal antibodies.