KEAP1 (D1G10) Rabbit mAb

7705:

Western Blotting

KEAP1 (D1G10) Rabbit mAb recognizes endogenous levels of total KEAP1 protein.

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly524 of human KEAP1 protein.

Western Blotting

Western blot analysis of extracts from various cell lines using KEAP1 (D1G10) Rabbit mAb.

Western Blotting

Western blot analysis of extracts from OVCAR8 cells, transfected with 100 nM SignalSilence^® Control siRNA (Unconjugated) #6568 (-), SignalSilence^® KEAP1 siRNA I #5285 (+) or SignalSilence^® KEAP1 siRNA II #5289 (+), using KEAP1 (D1G10) Rabbit mAb (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The KEAP1 (D1G10) Rabbit mAb confirms silencing of KEAP1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

The nuclear factor-like 2 (NRF2) transcriptional activator binds antioxidant response elements (ARE) of target gene promoter regions to regulate expression of oxidative stress response genes. Under basal conditions, the NRF2 inhibitor INrf2 (also called KEAP1) binds and retains NRF2 in the cytoplasm where it can be targeted for ubiquitin-mediated degradation (1). Small amounts of constitutive nuclear NRF2 maintains cellular homeostasis through regulation of basal expression of antioxidant response genes. Following oxidative or electrophilic stress, KEAP1 releases NRF2, thereby allowing the activator to translocate to the nucleus and bind to ARE-containing genes (2). The coordinated action of NRF2 and other transcription factors mediates the response to oxidative stress (3). Altered expression of NRF2 is associated with chronic obstructive pulmonary disease (COPD) (4). NRF2 activity in lung cancer cell lines directly correlates with cell proliferation rates, and inhibition of NRF2 expression by siRNA enhances anti-cancer drug-induced apoptosis (5).

KEAP1 contains an amino terminal BTB/POZ domain and a carboxyl terminal KELCH domain (6,7). The KELCH domain is required for interaction with NRF2, and the BTB/POZ domain functions in binding Cul3 E3 ubiquitin ligase (8-10). Under normal conditions, the complex leads to the cytoplasmic sequestration and ubiquitin-mediated proteasomal degradation of NRF2. Electrophilic modification of KEAP1 leads to disassociation of the NRF2/KEAP1 complex. KEAP1 also targets the down regulation of NF-κB activity by targeting IKKβ degradation (11). Mutation of the corresponding KEAP1 gene is seen in lung cancer cases and can lead to uncontrolled activation of NRF2 (12-14).

1. Cullinan, S.B. et al. (2004) Mol Cell Biol 24, 8477-86.
2. Nguyen, T. et al. (2005) J Biol Chem 280, 32485-92.
3. Jaiswal, A.K. (2004) Free Radic Biol Med 36, 1199-207.
4. Suzuki, M. et al. (2008) Am J Respir Cell Mol Biol 39, 673-82.
5. Homma, S. et al. (2009) Clin Cancer Res 15, 3423-32.
6. Itoh, K. et al. (1999) Genes Dev 13, 76-86.
7. Dhakshinamoorthy, S. and Jaiswal, A.K. (2001) Oncogene 20, 3906-17.
8. Furukawa, M. and Xiong, Y. (2005) Mol Cell Biol 25, 162-71.
9. Zhang, D.D. et al. (2004) Mol Cell Biol 24, 10941-53.
10. Kobayashi, A. et al. (2004) Mol Cell Biol 24, 7130-9.
11. Lee, D.F. et al. (2009) Mol Cell 36, 131-40.
12. Padmanabhan, B. et al. (2006) Mol Cell 21, 689-700.
13. Singh, A. et al. (2006) PLoS Med 3, e420.
14. Ohta, T. et al. (2008) Cancer Res 68, 1303-9.

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• 7003 20X LumiGLO^® Reagent and 20X Peroxide

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