The retinoblastoma susceptibility gene product: a characteristic pattern in normal cells and abnormal expression in...

Immunoprecipitation and Western immunoblotting studies were undertaken using purified high-affinity antibodies against a synthetic peptide corresponding to a portion of the deduced retinoblastoma (RB) protein. On SDS-PAGE, normal human cells showed an RB protein pattern consisting of a lower sharp band with a Mr of 110 kD and a more variable region above this band with a Mr ranging from 110 kD to 116 kD. The 110 kD band represents the unphosphorylated Rb protein whereas the broader, less well defined region is the phosphorylated RB protein in which molecular mass heterogenicity results from varying amount of phosphorylation. This pattern repeats once at a lower Mr in which a 98 kD band and 98-104 kD variable region can be visualized. This latter conformation seems to represent the unphosphorylated and phosphorylated RB protein translated from the second AUG codon of the RB mRNA. Cellular RB mRNA extracted from normal fibroblasts was translated in vitro reinforcing the usage of this second start codon. A higher ratio of phosphorylated to unphosphorylated Rb protein was seen in cells growing in log phase compared to those arrested in G1 phase. Our present studies also detected two candidates for RB-associated cellular proteins with a Mr of 124 kD and 55 kD respectively. In addition, shortened versions of RB-isoantigenic proteins were found in retinoblastoma and osteosarcoma cell lines.