Cell Signaling Technology

Product Pathways - Protein Stability

SignalSilence® UBE2S siRNA I #7220

Applications Reactivity
Transfection H (M) (R) (Mk)

Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
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 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® UBE2S siRNA I (+), or SignalSilence® UBE2S siRNA II #7732 (+), using UBE2S Antibody #9630 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The UBE2S Antibody confirms silencing of UBE2S expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.

Description

SignalSilence® UBE2S siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit UBE2S 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 from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

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 SignalSilence® UBE2S siRNA I 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

Protein ubiquitination requires the concerted action of the E1, E2, and E3 ubiquitin-conjugating enzymes. Ubiquitin is first activated through ATP-dependent formation of a thiol ester with ubiquitin-activating enzyme E1. The activated ubiquitin is then transferred to a thiol group of ubiquitin-carrier enzyme E2. The final step is the transfer of ubiquitin from E2 to an ε-amino group of the target protein lysine residue, which is mediated by ubiquitin-ligase enzyme E3 (1).

The human anaphase promoting complex (APC/C) is a large macromolecular E3 ligase complex that is largely responsible for the timely progression through mitosis via the sequential targeting of cell cycle regulators for proteasomal degradation. Recent work has revealed that APC/C substrates are marked for proteasomal degradation during cell cycle progression through the covalent assembly of Lys11-linked ubiquitin chains, which occurs through a priming phase and elongation phase (2-5). The APC/C utilizes, in part, the UBE2C/UBCH10 E2 enzyme to prime substrates for degradation through the covalent attachment of short Lys11-linked chains (3,6). The Lys11-specific elongating E2 enzyme, UBE2S/E2-EPF, extends these short chains into long Lys11-linked ubiquitin chains on APC/C bound substrates (2,3,7).In addition to the well-established biochemical role for UBE2S in cell cycle regulation, this enzyme has been shown to be overexpressed in many types of human cancer (8) and has also been implicated in hypoxia signaling (9,10). Indeed, UBE2S has been reported to associate with VHL and to target it for proteasomal degradation, thereby stabilizing HIF-1α (9).

  1. Hershko, A. (1988) J. Biol. Chem. 263, 15237-15240.
  2. Williamson, A. et al. (2009) Proc Natl Acad Sci USA 106, 18213-8.
  3. Jin, L. et al. (2008) Cell 133, 653-65.
  4. Wu, T. et al. (2010) Proc Natl Acad Sci USA 107, 1355-60.
  5. Song, L. and Rape, M. (2010) Mol Cell 38, 369-82.
  6. Summers, M.K. et al. (2008) Mol Cell 31, 544-56.
  7. Wickliffe, K.E. et al. (2011) Cell 144, 769-81.
  8. Tedesco, D. et al. (2007) Neoplasia 9, 601-13.
  9. Jung, C.R. et al. (2006) Nat Med 12, 809-16.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

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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