Cell Signaling Technology

Product Pathways - siRNA

SignalSilence® YB1 siRNA II #6207

Applications Reactivity
Transfection H

Reactivity Key:  H=Human
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® YB1 siRNA I #6206 (+) or SignalSilence® YB1 siRNA II (+), using YB1 Antibody #2749 and GAPDH (14C10) Rabbit mAb #2118. The YB1 antibody confirms silencing of YB1 expression, while the GAPDH (14C10) rabbit mAb was used to control for loading and specificity of YB1 siRNA.

Description

SignalSilence® YB1 siRNA from Cell Signaling Technology (CST) allows the researcher to specifically inhibit YB1 expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products are rigorously tested in-house and have been shown to reduce target protein expression in specified cell lines.

Quality Control

Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.

Directions for Use

CST recommends transfection with 100 nM YB1 siRNA II 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use.

Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.

Background

The Y-box binding protein 1 (YB1) belongs to a family of evolutionarily conserved, multifunctional Y-box proteins that bind single-stranded DNA and RNA and function as regulators of transcription, RNA metabolism, and protein synthesis (1). YB1 binds to Y-box sequences (TAACC) found in multiple gene promoters and can positively or negatively regulate transcription. YB1 activates genes associated with proliferation and cancer, such as cyclin A, cyclin B1, matrix metalloproteinase-2 (MMP-2), and the multi-drug resistance 1 (MDR1) gene (2-4). YB1 represses genes associated with cell death, including the Fas cell death-associated receptor and the p53 tumor suppressor gene (5-7). It also interacts with the RNA-splicing factor SRp30c and stabilizes interleukin-2 (IL-2) mRNA upon induction of T lymphocytes by IL-2 (8,9). The majority of YB1 protein localizes to the cytoplasm, with a minor pool found in the nucleus; however, nuclear localization appears to be critical for its role in promoting proliferation. Nuclear translocation is cell cycle regulated, with YB1 protein accumulating in the nucleus during G1/S phase (2). In addition, nuclear translocation is induced in response to extracellular stimuli such as hyperthermia and UV irradiation, or treatment of cells with thrombin, interferons, or insulin-like growth factor (IGF-I) (2,10). Treatment of the MCF7 breast cancer cell line with IGF-I results in Akt-mediated phosphorylation of YB1 at Ser102, which is required for nuclear translocation of YB1 and its ability to promote anchorage-independent growth (10). Research studies have shown that YB1 is overexpressed in many malignant tissues, including breast cancer, non-small cell lung carcinoma, ovarian adenocarcinomas, human osteosarcomas, colorectal carcinomas, and malignant melanomas. Investigators have shown that nuclear YB1 expression correlates with high levels of proliferation, drug resistance, and poor tumor prognosis (2,7,10).

  1. Matsumoto, K. and Wolffe, A.P. (1998) Trends Cell Biol. 8, 318-23.
  2. Jurchott, K. et al. (2003) J. Biol. Chem. 278, 27988-96.
  3. Mertens, P.R. et al. (1997) J. Biol. Chem. 272, 22905-12.
  4. Uchiumi, T. et al. (1993) Cell Growth Differ. 4, 147-57.
  5. Lasham, A. et al. (2000) Gene 252, 1-13.
  6. Lasham, A. et al. (2003) J. Biol. Chem. 278, 35516-23.
  7. Homer, C. et al. (2005) Oncogene 24, 8314-25.
  8. Raffetseder, U. et al. (2003) J. Biol. Chem. 278, 18241-8.
  9. Chen, C.Y. et al. (2000) Genes Dev. 14, 1236-48.
  10. Sutherland, B.W. et al. (2005) Oncogene 24, 4281-92.

Application References

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Companion Products

Limited Use Label License, RNA interference: This product is licensed under European Patent 1144623 and foreign equivalents from Ribopharma AG, Kulmbach, Germany and is provided only for use in non-commercial research specifically excluding use (a) in drug discovery or drug development, including target identification or target validation, by or on behalf of a commercial entity, (b) for contract research or commercial screening services, (c) for the production or manufacture of siRNA-related products for sale, or (d) for the generation of commercial databases for sale to Third Parties. Information about licenses for these and other commercial uses is available from Ribopharma AG, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Germany.

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

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