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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 (Ser780) (D59B7) Rabbit mAb.

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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).

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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.

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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.

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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.

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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.

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Western blot analysis of phosphorylated or nonphosphorylated recombinant, truncated Rb, without or with Rb blocking peptide, using Phospho-Rb (Ser780) (D59B7) Rabbit mAb.

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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.

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

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Immunoprecipitation of phospho-Rb (Ser780) from WI-38 cell extracts using Phospho-Rb (Ser780) (D59B7) Rabbit mAb (lane 2). Western blot was performed using the same antibody. Lane 1 is 10% input.

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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.

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

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

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

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

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

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

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

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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).

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Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 Raji cells and either 5 μl of Rb (4H1) Mouse 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.

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

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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 (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Image
Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Rb (Ser780) (D59B7) Rabbit mAb 8180 100 µl
H M R Mk 110 Rabbit IgG
Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb 8516 100 µl
H M R Mk 110 Rabbit IgG
Rb (4H1) Mouse mAb 9309 100 µl
H Mk B Pg 110 Mouse IgG2a
Rb Control Proteins 9303 100 µl
76  
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
All Goat 
Anti-biotin, HRP-linked Antibody 7075 100 µl
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
All Horse 
20X LumiGLO® Reagent and 20X Peroxide 7003 5 ml each
 
Biotinylated Protein Ladder Detection Pack 7727 100 µl
 

Product Description

The PhosphoPlus® Rb (Ser780, Ser807/811) Antibody Kit provides reagents and protocols to investigate cell cycle progression within cells. The kit contains a total Rb antibody, two distinct phospho-Rb antibodies, and Rb control proteins along with secondary antibodies and reagents to perform up to 10 western blots.


Specificity / Sensitivity

Phospho-Rb (Ser780) (D59B7) Rabbit mAb and Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb detect endogenous levels of Rb only when phosphorylated at Ser780 and Ser807/811, respectively. Rb (4H1) Mouse mAb detects endogenous levels of total Rb protein. The antibody does not cross-react with the Rb homologues p107 or p130, or with other proteins.


Source / Purification

Phospho-Rb (Ser780) (D59B7) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser780 of human Rb protein. Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser807/811 of human Rb protein. Rb (4H1) monoclonal antibody is produced by immunizing animals with Rb-C Fusion Protein #6022, which contains residues 701-928 of human Rb .

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.


Entrez-Gene Id 5925
Swiss-Prot Acc. P06400


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.
LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories.