Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Chromatin Regulation / Epigenetics

RNF20 (D6E10) XP® Rabbit mAb #11974

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP ChIP H M R Mk (Hm) (Dg) (Pg) (GP) (Hr) Endogenous 120 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  Dg=Dog  Pg=Pig  GP=Guinea Pig  Hr=Horse
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

RNF20 (D6E10) XP® Rabbit mAb recognizes endogenous levels of total RNF20 protein. This antibody recognizes a second band at 80 kDa in mouse lysates, corresponding to mouse RNF20 isoform 2. This antibody does not cross-react with RNF40 protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly516 of human RNF20 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using RNF20 (D6E10) XP® Rabbit mAb.

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 μl of RNF20 (D6E10) XP® Rabbit 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 SimpleChIP® Human γ-Actin Promoter Primers #5037, SimpleChIP® Human GAPDH Promoter Primers #4471, 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.

Background

In mammalian cells, the significance of histone H2B ubiquitination in chromatin epigenetics came from the identification of the budding yeast protein Bre1 (1,2). Together with the ubiquitin-conjugating enzyme Rad6, Bre1 serves as the E3 ligase in the monoubiquitination of the yeast histone H2B within transcribed regions of chromatin (1-3). Subsequently, the mammalian orthologs of yeast Bre1, RNF20 and RNF40, were identified (4,5). These two proteins form a tight heterodimer that acts as the major E3 ligase responsible for histone H2B monoubiquitination at Lys120 in mammalian cells, a modification linked to RNA Pol II-dependent transcription elongation in undamaged cells. Researchers have shown that DNA double-strand breaks (DSBs) are also capable of inducing monoubiquitination of H2B. This process depends upon the recruitment to DSB sites, as well as ATM-dependent phosphorylation of the RNF20-RNF40 heterodimer, thus highlighting a role for this E3 ligase in DSB repair pathways (6). Indeed, investigators have shown that loss of RNF20-RNF40 function promotes replication stress and chromosomal instability, which may constitute an early step in malignant transformation that precedes cell invasion (7).

  1. Wood, A. et al. (2003) Mol Cell 11, 267-74.
  2. Hwang, W.W. et al. (2003) Mol Cell 11, 261-6.
  3. Kao, C.F. et al. (2004) Genes Dev 18, 184-95.
  4. Kim, J. et al. (2005) Mol Cell 20, 759-70.
  5. Zhu, B. et al. (2005) Mol Cell 20, 601-11.
  6. Moyal, L. et al. (2011) Mol Cell 41, 529-42.
  7. Chernikova, S.B. et al. (2012) Cancer Res, Epub ahead of print.

Application References

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Companion Products

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

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