Product Pathways - Cell Cycle / Checkpoint

Cell Cycle Regulation Sampler Kit #9932

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

Specificity / Sensitivity

Antibodies detect endogenous levels of their respective proteins.

Western Blotting

Western blot analysis of whole cell extracts from C6, NIH/3T3, HeLa and MCF-7 cells using p27 Kip1 Antibody #2552.

Western Blotting

Western blot analysis of SK-N-MC and IMCD3 cell lysates using CDK4 (DCS156) mAb #2906.

Western Blotting

Western blot analysis of extracts from HeLa and NIH/3T3 cells using Cyclin D1 (DCS) mAb #2926.

Western Blotting

Western blot analysis of extracts from SK-N-MC, C6 and IMCD3 cells using Cyclin D3 (DCS22) mAb #2936.

Western Blotting

Western blot analysis of whole cell lysates from 293, MCF-7 and COS cells using p21 Waf1/Cip1 (DCS60) mAb #2946.

Western Blotting

Western blot analysis of extracts from HeLa, IM-CD-3 and C6 cell lysates using CDK6 (DCS83) mAb #3136.

Western Blotting

Western blot analysis of whole cell extracts from 293, HeLa, HT29, C6 and NIH/3T3 cells using p15 INK4B Antibody #4822.

Western Blotting

Western blot analysis of extracts from HeLa and SK-N-MC cells using p16 INK4A Antibody #4824.

IHC-P (paraffin)

Immunohistochemical staining of cyclin D1 in paraffin-embedded human breast carcinoma showing nuclear localization, using Cyclin D1 (DCS6) mAb #2926.

IHC-P (paraffin)

Immunohistochemical staining of cyclin D3 in paraffin-embedded human breast carcinoma showing nuclear localization, using Cyclin D3 (DCS22) mAb #2936.

IHC-P (paraffin)

Immunohistochemical staining of p21 Waf1/Cip1 in paraffin-embedded human breast carcinoma showing nuclear localization, using p21 Waf1/Cip1 (DCS60) mAb #2946.

Flow Cytometry

Flow cytometric analysis of untreated C6 cells, using p15 INK4B Antibody # 4822 versus propidium iodide (DNA content). The boxed population indicates p15 INK4B-positive cells.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic peptides (KLH-coupled) and are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing mice with recombinant human preoteins.

Background

Eukaryotic cell cycle progression is dependent, in part, on the tightly regulated activity of cyclin dependent kinases (CDKs). Cyclin D/CDK4/6 activity occurs in mid-late G1 phase, upstream of CDK2/cyclin E activity. Both of these activities are required for hyperphosphorylation of the retinoblastoma gene product (pRb). pRb phosphorylation allows the release of S phase-promoting transcription factors and is indicative of the cell's commitment to proliferate. This point in the cell cycle is known as the restriction point. Cyclin protein levels oscillate throughout the cell cycle, and their availability is a means of controlling CDK activity and cell proliferation. Cyclin D is degraded through the ubiquitin proteasome pathway in the absence of mitogenic signaling. Ubiquitination of cyclin D1 is enhanced by phosphorylation at Thr286 by glycogen synthase kinase 3b (GSK-3b) (1). p27/Kip1, p57 Kip2 and p21 Waf1/Cip1 are members of the Cip/Kip family of cyclin-dependent kinase inhibitors. They form heterotrimeric complexes with cyclins and CDKs, inhibiting kinase activity and blocking progression through G1/S phase (2). However, p21 may enhance assembly and activity of cyclin D/CDK4/6 complexes (3). Levels of p21 and p27 protein are controlled through ubiquitination and proteasomal degradation (4). Levels of p27 are upregulated in quiescent cells and in cells treated with negative cell cycle regulators. p27 nuclear localization is controlled by Akt-dependent phosphorylation at Thr157 (5). The inhibitors of CDK4 (INK4) family include p15 INK4B, p16 INK4A, p18 INK4C, and p19 INK4D. All INK4 proteins selectively inhibit CDK4/6 activity, either in a binary complex, or in a ternary complex including cyclin D, resulting in inhibition of cell division (6,7).

1. Diehl, J.A. et al. (1997) Genes Dev. 11, 957-972.
2. Pestell, R.G. et al. (1999) Endocrine Rev. 20, 501-534.
3. Cheng, J. et al. (1999) EMBO J. 18, 1571-1573.
4. Sheaff, R.J. et al. (2000) Cell 5, 403-410.
5. Shin, I. et al. (2002) Nat. Med. 8, 1145-1152.
6. Guan, K.L. et al. (1994) Genes Dev. 8, 2939-2952.
7. Hirai, H. et al. (1995) Mol. Cell. Biol. 15, 2672-2681.

Application References

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