Revision 6

#21792Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IHC-P, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

Iso1 60, Iso2 50

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q15532

Entrez-Gene Id:

6760

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:10000
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

SS18 (D6I4Z) Rabbit mAb recognizes endogenous levels of total SS18 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Dog, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln394 of human SS18 protein.

Background

ATP-dependent chromatin remodeling complexes play an essential role in the regulation of nuclear processes such as transcription and DNA replication and repair (1,2). The SWI/SNF chromatin remodeling complex consists of more than 10 subunits and contains a single molecule of either BRM or BRG1 as the ATPase catalytic subunit. The activity of the ATPase subunit disrupts histone-DNA contacts and changes the accessibility of crucial regulatory elements to the chromatin. The additional core and accessory subunits play a scaffolding role to maintain stability and provide surfaces for interaction with various transcription factors and chromatin (2-5). The interactions between SWI/SNF subunits and transcription factors, such as nuclear receptors, p53, Rb, BRCA1, and MyoD, facilitate recruitment of the complex to target genes for regulation of gene activation, cell growth, cell cycle, and differentiation processes (1,6-9).
SS18 is a protein that has been shown to be a part of the SWI/SNF complex (10, 11). The SS18-SSX fusion proteins are a result of in-frame fusions that fuse the SS18 gene on chromosome 18 with X chromosome genes SSX1, SSX2, and to a lesser extent SSX4 (12). Human synovial sarcoma (SS) accounts for 8-10% of all soft tissue malignancies and 95% of these malignancies express the recurrent translocation of the SS18 gene on chromosome 18 (12). The N-terminal SNH domain (SYT N-terminal homology domain) of the SS18 protein interacts with SWI/SNF chromatin remodeling complexes via the N terminal region of BRM and BRG1 subunits (13). Studies of the SS18-SSX fusion in SS suggest that endogenous SS18 competes with the mutant SS18-SSX fusion for occupancy in the SWI/SNF complexes resulting in the displacement of SNF5 (BAF47) subunit. Displacement of the SNF5 subunit results in altered function of the SWI/SNF complex that leads to deregulated expression of genes such as Sox2 in SS (12).
In addition, cytosolic SS18 isoforms also associate with F-actin in cytoskeletal organization (14). SS18 null mice do not develop beyond E9.5 and have defects in vascularization, cell migration, neural tube closure, and fusion within the embryonic-maternal membranes (14).

  1. Ho, L. and Crabtree, G.R. (2010) Nature 463, 474-84.
  2. Becker, P.B. and Hörz, W. (2002) Annu Rev Biochem 71, 247-73.
  3. Eberharter, A. and Becker, P.B. (2004) J Cell Sci 117, 3707-11.
  4. Bowman, G.D. (2010) Curr Opin Struct Biol 20, 73-81.
  5. Gangaraju, V.K. and Bartholomew, B. (2007) Mutat Res 618, 3-17.
  6. Lessard, J.A. and Crabtree, G.R. (2010) Annu Rev Cell Dev Biol 26, 503-32.
  7. Morettini, S. et al. (2008) Front Biosci 13, 5522-32.
  8. Wolf, I.M. et al. (2008) J Cell Biochem 104, 1580-6.
  9. Simone, C. (2006) J Cell Physiol 207, 309-14.
  10. Nagai, M. et al. (2001) Proc Natl Acad Sci U S A 98, 3843-8.
  11. Thaete, C. et al. (1999) Hum Mol Genet 8, 585-91.
  12. Kadoch, C. and Crabtree, G.R. (2013) Cell 153, 71-85.
  13. Perani, M. et al. (2003) Oncogene 22, 8156-67.
  14. Kim, J. et al. (2009) PLoS One 4, e6455.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 6
#21792

SS18 (D6I4Z) Rabbit mAb

Western Blotting Image 1: SS18 (D6I4Z) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using SS18 (D6I4Z) Rabbit mAb.
Immunoprecipitation Image 1: SS18 (D6I4Z) Rabbit mAb Expand Image
Immunoprecipitation of SS18 from ACHN cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype control #3900, and lane 3 is SS18 (D6I4Z) Rabbit mAb. Western blot analysis was performed using SS18 (D6I4Z) Rabbit mAb.
Immunohistochemistry Image 1: SS18 (D6I4Z) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using SS18 (D6I4Z) Rabbit mAb (left) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemistry Image 2: SS18 (D6I4Z) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using SS18 (D6I4Z) Rabbit mAb.
Immunohistochemistry Image 3: SS18 (D6I4Z) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using SS18 (D6I4Z) Rabbit mAb.
Immunohistochemistry Image 4: SS18 (D6I4Z) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using SS18 (D6I4Z) Rabbit mAb.
Immunohistochemistry Image 5: SS18 (D6I4Z) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human synovial sarcoma using SS18 (D6I4Z) Rabbit mAb.
Chromatin Immunoprecipitation Image 1: SS18 (D6I4Z) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d, followed by treatment with β-estradiol (10 nM, 45 min) and either SMARCC1/BAF155 (D7F8S) Rabbit mAb #11956, SMARCB1/BAF47 (D8M1X) Rabbit mAb #91735, or SS18 (D6I4Z) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. SMARCC1/BAF155, SMARCB1/BAF47, and SS18 are all subunits of SWI/SNF complex. The figure shows binding across pS2/TFF1, a known target gene of SWI/SNF complex (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: SS18 (D6I4Z) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d, followed by treatment with β-estradiol (10 nM, 45 min) and either SMARCC1/BAF155 (D7F8S) Rabbit mAb #11956, SMARCB1/BAF47 (D8M1X) Rabbit mAb #91735, or SS18 (D6I4Z) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. SMARCC1/BAF155, SMARCB1/BAF47, and SS18 are all subunits of SWI/SNF complex. The figure shows binding across chromosome 21 (upper), including pS2/TFF1 (lower), a known target gene of SWI/SNF complex (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: SS18 (D6I4Z) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d then treated with β-estradiol (10 nM) for 45 min and either SS18 (D6I4Z) Rabbit mAb or 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 pS2 Promoter Primers #9702, 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.
CUT and RUN Image 1: SS18 (D6I4Z) Rabbit mAb Expand Image
CUT&RUN was performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d then treated with β-estradiol (10 nM) for 45 min and SS18 (D6I4Z) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across DEPTOR, a known target gene of SS18 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: SS18 (D6I4Z) Rabbit mAb Expand Image
CUT&RUN was performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d then treated with β-estradiol (10 nM) for 45 min and SS18 (D6I4Z) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 8 (upper), including DEPTOR (lower), a known target gene of SS18 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: SS18 (D6I4Z) Rabbit mAb Expand Image
CUT&RUN was performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d, followed by treatment with β-estradiol (10 nM, 45 min) and either SS18 (D6I4Z) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human HMCN1 exon 96 primers, human TFF1/pS2 promoter primers, human DEPTOR intron 4 primers, and ITM2A upstream primers. 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.