Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

Rb (D20) Rabbit mAb #9313

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP ChIP H M Mk Endogenous 110 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Rb (D20) Rabbit mAb detects endogenous levels of total Rb protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to central residues of mouse Rb.

Western Blotting

Western Blotting

Western blot analysis of extracts from WI-38 cells, serum-starved for three days and serum-stimulated for the indicated times, using Rb (D20) Rabbit mAb.

IP

IP

Immunoprecipitation of Rb from Jurkat cells using Rb (D20) Rabbit mAb. Western blot was performed using the same antibody.

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 Raji cells and either 5 μl of Rb (D20) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using using SimpleChIP® Human Timeless Intron 1 Primers #7001, human DHFR promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.


Background

The retinoblastoma tumor suppressor protein, Rb, regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).

  1. Sherr, C.J. (1996) Science 274, 1672-7.
  2. Nevins, J.R. (1992) Science 258, 424-9.
  3. Welch, P.J. and Wang, J.Y. (1993) Cell 75, 779-90.
  4. Hu, Q.J. et al. (1990) EMBO J 9, 1147-55.
  5. Knudsen, E.S. and Wang, J.Y. (1997) Mol Cell Biol 17, 5771-83.
  6. Lundberg, A.S. and Weinberg, R.A. (1998) Mol Cell Biol 18, 753-61.
  7. Connell-Crowley, L. et al. (1997) Mol Biol Cell 8, 287-301.
  8. Kitagawa, M. et al. (1996) EMBO J 15, 7060-9.
  9. Geng, Y. et al. (2001) Proc Natl Acad Sci USA 98, 194-9.

Application References

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Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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