Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

SIN3A (D9D6) Rabbit mAb #8056

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC ChIP H Mk Endogenous 145 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

SIN3A (D9D6) Rabbit mAb recognizes endogenous levels of total SIN3A protein. Based on protein sequence, this antibody is not predicted to cross-react with SIN3B protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu530 of human SIN3A protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using SIN3A (D9D6) Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of 293 cells using SIN3A (D9D6) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 MCF7 cells grown in phenol red free medium and charcoal stripped FBS for 4 days then treated with β-estradiol (10 nM) for 1 hr and either 10 μl of SIN3A (D9D6) 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 ESR1 Promoter Primers #9673, SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human MyoD Exon 1 Primers #4490, 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

SIN3 was originally identified as a negative regulator of transcription in budding yeast (1,2). Since then, three isoforms of the SIN3 proteins have been identified in mammalian cells, as products of two different genes, SIN3A and SIN3B (3,4). Both SIN3A and SIN3B are nuclear proteins that function as scaffolding subunits for the multi-subunit SIN3 transcriptional repressor complex, containing SIN3A or SIN3B, HDAC1, HDAC2, SDS3, RBBP4/RBAP48, RBBP7/RBAP46, SAP30, and SAP18 (3,4). SIN3 proteins contain four paired amphipathic alpha-helix (PAH) motifs that function in the recruitment of the SIN3 complex to target genes by binding a multitude of DNA-binding transcriptional repressor proteins, including Mad1, p53, E2F4, HCF-1, AML1, Elk-1, NRSF, CTCF, ERα, and MeCP2 (3,4). In addition, SIN3 proteins contain an HDAC interaction domain (HID), which mediates binding of HDAC1 and HDAC2 via the SDS3 bridging protein, and a highly conserved region (HCR) at the carboxy terminus, which contributes to repressor protein binding (3,4). RBBP4 and RBBP7 proteins also bind to SDS3 and contribute to nucleosome binding of the complex. The SIN3 complex functions to repress transcription, in part, by deacetylating histones at target gene promoters (3,4). In addition, recent studies have shown that SIN3 is recruited to the coding regions of repressed and active genes, where it deacetylates histones and suppresses spurious transcription by RNA polymerase II (3,5). In addition to histone deacetylase activity, the SIN3 complex associates with histone methyltransferase (ESET), histone demethylase (JARID1A/RBP2), ATP-dependent chromatin remodeling (SWI/SNF), methylcytosine dioxygenase (TET1), and O-GlcNAc transferase (OGT) activities, all of which appear to contribute to the regulation of target genes (5-9). The SIN3 complex is critical for proper regulation of embryonic development, cell growth and proliferation, apoptosis, DNA replication, DNA repair, and DNA methylation (imprinting and X-chromosome inactivation) (3,4).

  1. Sternberg, P.W. et al. (1987) Cell 48, 567-77.
  2. Nasmyth, K. et al. (1987) Cell 48, 579-87.
  3. Grzenda, A. et al. Biochim Biophys Acta 1789, 443-50.
  4. McDonel, P. et al. (2009) Int J Biochem Cell Biol 41, 108-16.
  5. van Oevelen, C. et al. (2008) Mol Cell 32, 359-70.
  6. Yang, L. et al. (2003) Biochem J 369, 651-7.
  7. Sif, S. et al. (2001) Genes Dev 15, 603-18.
  8. Williams, K. et al. (2011) Nature 473, 343-8.
  9. Yang, X. et al. (2002) Cell 110, 69-80.

Application References

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

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