LOSS OF IMMUNOSTAINING OF THE RBP-J(KAPPA) TRANSCRIPTION FACTOR UPON F9 CELL-DIFFERENTIATION INDUCED BY RETINOIC ACID

RBP-J(kappa) is a novel type of transcriptional regulatory protein tha t does not contain any known DNA-binding motif, We raised anti-RBP-J(k appa) monoclonal antibodies (K0043 and T6709) to investigate the roles of RBP-J(kappa) in cell differentiation, These antibodies stained nuc lei of undifferentiated embryonic stem cells and F9 cells but not thos e of the other differentiated cell lines tested so far although the RB P-J(kappa) protein is expressed at similar levels, Interestingly, diff erentiated F9 cells lost the immunostaining reaction with the anti-RBP -J(kappa) monoclonal antibodies, Biochemical subcellular fractionation study showed that the majority of REP-J(kappa) was localized in nucle i of F9 cells and that there are at least two forms of the RBP-J(kappa ) protein in the nuclei of undifferentiated F9 cells, a free form and a chromatin-bound form, Upon induction of F9 cell differentiation, fre e nuclear RBP-J(kappa) disappeared concomitantly with the loss of immu nostaining, suggesting that the anti-RBPJ(kappa) antibodies cannot rec ognize chromatin-bound RBP-J(kappa). Since there is no evidence to ind icate covalent modification of RBP-J(kappa), we assume that chromatin- bound RBP-J(kappa) interacts with a large number of proteins which blo ck the exposure of RBP-J kappa epitopes to the monoclonal antibodies.