Cell Signaling Technology

Product Pathways - Protein Stability

SignalSilence® UBA2 siRNA II #7642

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 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® UBA2 siRNA I #7646 (+) or SignalSilence® UBA2 siRNA II (+), using UBA2 (D15C11) Rabbit mAb #8688 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The UBA2 (D15C11) Rabbit mAb confirms silencing of UBA2 expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.

Description

SignalSilence® UBA2 siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit UBA2 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® UBA2 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 process of SUMO conjugation to target proteins is similar to the molecular chain of events observed with ubiquitin (1). SUMO is conjugated to target proteins through the coordinated action of the cellular SUMO conjugation machinery consisting of E1, E2, and E3 enzymes (2). The canonical SUMO E1 activating enzyme is a heterodimer consisting of SAE1 (AOS1) and UBA2 (SAE2) subunits. Mature SUMO is activated by E1 in an ATP-dependent reaction that generates adenylated SUMO, which functions as a high-energy intermediate in the formation of a thioester linkage between SUMO and Cys173 of UBA2 (3,4). SUMO is subsequently transferred from UBA2 to the SUMO E2 conjugating enzyme, UBC9 (5). Recent evidence suggests that redox regulation of UBA2 serves as a physiologic mechanism to modulate the cellular level of sumoylated target proteins (6).

  1. Geiss-Friedlander, R. and Melchior, F. (2007) Nat Rev Mol Cell Biol 8, 947-56.
  2. Tatham, M.H. et al. (2003) Biochemistry 42, 9959-69.
  3. Desterro, J.M. et al. (1999) J Biol Chem 274, 10618-24.
  4. Gong, L. et al. (1999) FEBS Lett 448, 185-9.
  5. Desterro, J.M. et al. (1997) FEBS Lett 417, 297-300.
  6. Bossis, G. and Melchior, F. (2006) Mol Cell 21, 349-57.

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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