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9300
PhosphoPlus® Rb (Ser780, Ser795, Ser807/811) Antibody Kit
Primary Antibodies

PhosphoPlus® Rb (Ser780, Ser795, Ser807/811) Antibody Kit #9300

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Western blot analysis of extracts from human fibroblasts synchronized by serum deprivation, using Phospho-Rb (Ser795) Antibody. Cells were synchronized for 24 hours, then released by addition of serum and harvested at the times indicated. Cell cycle progression was verified by cyclin analysis and FACS. (Provided by John Boylan, Dupont/Merck, Delaware.)

Western blot analysis of Rb-C Fusion Protein #6022 (amino acids 701-928 of Rb fused to MBP) before (-) and after (+) in vitro phosphorylation by cdc2/cyclin B Protein Kinase (New England Biolabs #P6020), using Phospho-Rb (Ser795) Antibody #9301 (left) or the control antibody (right).

Western blot analysis of extracts from human fibroblasts synchronized by serum deprivation, using Phospho-Rb (Ser780) Antibody. Cells were synchronized for 24 hours then released by addition of serum and harvested at the times indicated. Cell cycle progression was verified by cyclin analysis and FACS. (Provided by John Boylan, Dupont/Merck, Delaware.)

Western blot analysis of extracts from human fibroblasts synchronized by serum deprivation, using Phospho-Rb (Ser807/811) Antibody. Cells were synchronized for 24 hours then released by addition of serum and harvested at the times indicated. Cell cycle progression was verified by cyclin analysis and FACS. (Provided by John Boylan, Dupont/Merck, Delaware.)

Chromatin immunoprecipitations were performed with cross-linked chromatin from Raji cells and either Rb (4H1) Mouse mAb or 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.

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

Western blot analysis of Rb Control Protein #9303, using Phospho-Rb (Ser795) Antibody (upper) or Rb (4H1) mAb #9309 (lower).

Flow cytometric analysis of Jurkat cells, using Rb (4H1) Mouse mAb versus propidium iodide (DNA content). The box indicates Rb positive cells.

Confocal immunofluorescent image of SH-SY5Y cells, using RB (4H1) Mouse mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing nuclear localization, using Rb (4H1) Mouse mAb.

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Rb (4H1) Mouse mAb.

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-), SignalSilence® Rb siRNA I #6451 (+) or SignalSilence® Rb siRNA II (+), using Rb (4H1) Mouse mAb #9309 and α-Tubulin (11H10) Rabbit mAb #2125. The Rb (4H1) Mouse mAb confirms silencing of Rb expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of Rb siRNA.

Western blot analysis of extracts from COS-7 cells, untreated or hydroxyurea-treated (G1/S), using Rb (4H1) Mouse mAb.

To Purchase # 9300

Product Description

The PhosphoPlus® Rb (Ser780, Ser795, Ser807/811) Antibody Kit provides reagents and protocols for the rapid analysis of Rb phosphorylation.

Specificity / Sensitivity

Phospho-Rb (Ser780, Ser795, Ser807/811) Antibodies detect endogenous levels of Rb only when phosphorylated at the target sites. The monoclonal Rb antibody does not recognize the Rb homologues p107 or p130, or other proteins.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Ser780, Ser795 or Ser807/811 of human Rb. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a fusion protein (Rb-C Fusion Protein #6022) containing residues 701-928 of human Rb .

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.