Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Cell Cycle / Checkpoint

Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb #8516

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IF-IC F H M R Mk Endogenous 110 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  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

Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb recognizes endogenous levels of Rb protein only when phosphorylated at Ser807, Ser811, or at both sites. This antibody does not cross-react with Rb phosphorylated at Ser608.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser807/811 of human Rb protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with calf intestinal phosphatase (CIP) and λ phosphatase (+), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (upper) or Rb (4H1) Mouse mAb #9309 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from WI-38 cells, serum-starved for 3 days (-) or serum-starved for 3 days followed by treatment with 10% serum for 2 days (+), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

IP

IP

Immunoprecipitation of phospho-Rb (Ser807/811) from MCF7 cell extracts using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (lane 2). Western blot was performed using the same antibody. Lane 1 is 10% input.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded mouse spleen using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human ovarian serous adenocarcinoma using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb compared to Propidium Iodide (PI)/RNase Staining Solution #4087.

IF-IC

IF-IC

Confocal immunofluorescent analysis of MCF7 (left) and BT-549 (right) cells, untreated (upper) or λ phosphatase-treated (lower) using Phospho-Rb (Ser807/Ser811) (D20B12) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).


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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