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9917
Cell Cycle/Checkpoint Antibody Sampler Kit
Primary Antibodies

Cell Cycle/Checkpoint Antibody Sampler Kit #9917

Western Blotting Image 1

Western blot analysis of extracts from C6 cells, untreated (-) or treated with Nocodazole (0.1 μg/ml, 18 hr; +), using Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb (upper) or cdc2 Antibody #77055 (lower).

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Western Blotting Image 2

Western blot analysis of extracts from HeLa, COS, NIH/3T3 and C6 cells, untreated or UV-treated, using Phospho-Chk1 (Ser345) (133D30) Rabbit mAb.

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Western Blotting Image 3

Western blot analysis of extracts from HeLa cells, untreated or UV-treated, using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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Western Blotting Image 4

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 Blotting Image 5

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.)

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Western Blotting Image 6

Western blot analysis of extracts from HT29 cells, untreated or UV-treated (100 mJ/cm2, 1 hr), using Phospho-p53 (Ser15) (16G8) Mouse mAb (upper) or p53 (DO-7) Mouse mAb #48818 (lower).

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Western Blotting Image 7

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 Blotting Image 8

Western blot analysis of extracts from HeLa cells, untreated or hydroxyurea treated for 20 hours, using Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb.

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Flow Cytometry Image 9

Flow cytometric analysis of HeLa cells, untreated (blue) and UV-treated (green), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb.

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IP Image 10

Immunoprecipitation of phospho-chk2 from UV-treated HT29 cells using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb followed by western blot using the same antibody.

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Western Blotting Image 11

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 Blotting Image 12

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

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Flow Cytometry Image 13

Flow cytometric analysis of HT-29 cells, untreated (blue) or UV-treated (green), using Phospho-p53 (Ser15) (16G8) Mouse mAb compared to a nonspecific negative control antibody (red).

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Flow Cytometry Image 14

Flow cytometric analysis of Jurkat cells, using Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb versus propidium iodide (DNA content).

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IF-IC Image 15

Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or UV-treated (right), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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IP Image 17

Immunoprecipitation of phospho-Rb (Ser807/811) from Cos cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

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IF-IC Image 18

Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or UV-treated (right), using Phospho-p53 (Ser15) (16G8) Mouse mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

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IF-IC Image 19

Confocal immunofluorescent analysis of asynchronous HeLa cells labeled with Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb (green) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (red).

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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IHC-P (paraffin) Image 21

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

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IHC-P (paraffin) Image 22

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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IHC-P (paraffin) Image 23

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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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded HT-29 cell pellets, control (left) or UV-treated (right), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

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IHC-P (paraffin) Image 25

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

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Flow Cytometry Image 26

Flow cytometric analysis of untreated Jurkat cells, using Phospho-Chk2 (Thr68) (C13C1) Rb mAb versus propidium iodide (DNA content). The boxed population indicates phospho-Chk2 (Thr68)-positive cells.

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IHC-P (paraffin) Image 27

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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Flow Cytometry Image 28

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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IF-IC Image 29

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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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb 4539 20 µl
  • WB
  • IP
  • IF
  • F
H M R Mk 34 Rabbit 
Phospho-Chk1 (Ser345) (133D3) Rabbit mAb 2348 20 µl
  • WB
  • IF
  • F
H M R Mk 56 Rabbit IgG
Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb 2197 20 µl
  • WB
  • IP
  • IHC
  • F
H 62 Rabbit IgG
Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb 8516 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk 110 Rabbit IgG
Phospho-Rb (Ser795) Antibody 9301 20 µl
  • WB
  • IP
H R Mk 110 Rabbit 
Phospho-p53 (Ser15) (16G8) Mouse mAb 9286 20 µl
  • WB
  • IF
  • F
H 53 Mouse IgG1
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

The Cell Cycle/Checkpoint Antibody Sampler Kit provides a fast and economical means of evaluating multiple proteins involved in the cell cyle and checkpoint control. The kit contains enough primary and secondary antibody to perform four Western blot experiments.

Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb detects endogenous levels of cdc2 protein only when phosphorylated at tyrosine 15. Based on sequence similarity, the antibody may cross-react with CDK2 and CDK3. Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb detects endogenous levels of Chk2 only when phosphorylated at Thr68. Phospho-Chk1 (Ser345) Antibody detects Chk1 only when phosphorylated at Ser345 and does not cross-react with other proteins. Phospho-Rb (Ser795) Antibody detects Rb only when phosphorylated at Ser795 and does not cross-react with Rb phosphorylated at other sites. 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. Phospho-p53 (Ser15) (16G8) Mouse mAb detects endogenous levels of p53 only when phosphorylated at Ser15. The antibody does not cross-react with p53 phosphorylated at other sites.

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser795 of human Rb. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser807/811 of human Rb protein, residues surrounding Ser345 of human Chk1, residues surrounding Ser15 of human p53, residues surrounding Tyr15 of human cdc2, and residues surrounding Thr68 of human Chk2.

The cell division cycle demands accuracy to avoid the accumulation of genetic damage. This process is controlled by molecular circuits called "checkpoints" that are common to all eukaryotic cells (1). Checkpoints monitor DNA integrity and cell growth prior to replication and division at the G1/S and G2/M transitions, respectively. The cdc2-cyclin B kinase is pivotal in regulating the G2/M transition (2,3). Cdc2 is phosphorylated at Thr14 and Tyr15 during G2-phase by the kinases Wee1 and Myt1, rendering it inactive. The tumor suppressor protein retinoblastoma (Rb) controls progression through the late G1 restriction point (R) and is a major regulator of the G1/S transition (4). During early and mid G1-phase, Rb binds to and represses the transcription factor E2F (5). The phosphorylation of Rb late in G1-phase by CDKs induces Rb to dissociate from E2F, permitting the transcription of S-phase-promoting genes. In vitro, Rb can be phosphorylated at multiple sites by cdc2, cdk2, and cdk4/6 (6-8). DNA damage triggers both the G2/M and the G1/S checkpoints. DNA damage activates the DNA-PK/ATM/ATR kinases, which phosphorylate Chk at Ser345 (9), Chk2 at Thr68 (10) and p53 (11). The Chk kinases inactivate cdc25 via phosphorylation at Ser216, blocking the activation of cdc2.

  1. Nurse, P. (1997) Cell 91, 865-7.
  2. Norbury, C. and Nurse, P. (1992) Annu Rev Biochem 61, 441-70.
  3. Watanabe, N. et al. (1995) EMBO J. 14, 1878-1891.
  4. Sherr, C.J. (1996) Science 274, 1672-7.
  5. Dyson, N. (1998) Genes Dev 12, 2245-62.
  6. Kitagawa, M. et al. (1996) EMBO J 15, 7060-9.
  7. Lundberg, A.S. and Weinberg, R.A. (1998) Mol Cell Biol 18, 753-61.
  8. Harbour, J.W. et al. (1999) Cell 98, 859-69.
  9. Zhao, H. and Piwnica-Worms, H. (2001) Mol Cell Biol 21, 4129-39.
  10. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
  11. Tibbetts, R.S. et al. (1999) Genes Dev 13, 152-7.
Entrez-Gene Id
983 , 1111 , 11200 , 7157 , 5925
Swiss-Prot Acc.
P06493 , O14757 , O96017 , P04637 , P06400
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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