ppRB.sup.110 -phosphoprotein the retinoblastoma susceptibility gene product

DETAILED DESCRIPTION OF FIGURES FIG.
1 is the chromatogram illustrating the identification of RB proteins by immunoprecipitation with rabbit anti-RB IgG in various cell lines.
Human cells such as neuroblastoma LAN-1 (Lanes 1 and 2), Alexander hepatoma (Lane 3), osteosarcoma U20S (Lane 4), normal fibroblasts (Lane 5), and five retinoblastomas (Lanes 6 to 10) were labeled with .
sup.
35 S-methionine and immunoprecipitated with preimmune rabbit IgG (Lane 1) or rabbit anti-RB IgG (Lanes 2-10).
The immunoprecipitates were analyzed by 7.
5% SDS-polyacrylamide gel electrophoresis and autoradiographed.
FIG.
2 is the complete RB cDNA nucleotide sequences and predicted amino acid sequences of the RB protein.
The most 5' .
about.
240 nucleotides were obtained from a cDNA clone from retinoblastoma cell line Y79.
Nucleotide sequences from this clone and the original normal RB clones were aligned by sequence overlap.
The first and second initiation sites are boxed, and alanine and proline clusters underlined.
FIG.
3 is the chromatogram illustrating the modifications of the RB protein.
LAN-1 cells were labeled with .
sup.
35 S-methionine (lanes 1-3) or .
sup.
32 P-phosphoric acid (0.
5 mci/ml) (lanes 4 and 5) for three hours.
Cellular lysates were immunoprecipitated with preimmune rabbit IgG (lane 1 and 4) or anti-RB IgG (lanes 2, 3 and 5).
Aliquots of .
sup.
35 S-methionine-labeled RB proteins were digested with Endoglycosidase H (ICN ImmunoBiologicals) overnight (lane 3).
These immunoprecipitates were then analyzed by 7.
5% SDS-polyacrylamide gel as described in FIG.
1.
The RB gene product was found to be phosphorylated but not glycosylated.
FIG.
4 is the chromatogram illustrating a conservation of the RB gene product in different vertebrate species.
Cell lines of human neuroblastoma, LAN-5, (Lanes 1 and 2), monkey, cos, (lanes 3 and 4), quail fibroblast, QT6, (Lanes 5 and 6), mouse fibroblast, NIH/3T3, (Lanes 7 and 8), and rat fibroblast, rat-2, (Lanes 9 and 10) were labeled with .
sup.
32 P-phosphoric acid and immunoprecipitated with preimmune IgG (odd numbered lanes) or anti-RB IgG (even numbered lanes) and analyzed as described in FIG