Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

SignalSilence® ATR siRNA I #6288

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 UV treated HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® ATR siRNA I (+), using Phospho-(Ser/Thr) ATM/ATR Substrate (4F7) Rabbit mAb #2909 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Phospho-(Ser/Thr) ATM/ATR Substrate (4F7) Rabbit mAb confirms reduction of ATR kinase activity, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® ATR siRNA I (+) or SignalSilence® ATR siRNA II #6289 (+), using ATR Antibody #2790 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The ATR Antibody confirms silencing of ATR expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

SignalSilence® ATR siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit ATR 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 ATR 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

Ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) are PI3 Kinase-related kinase (PIKK) family members that phosphorylate multiple substrates on serine or threonine residues that are followed by a glutamine in response to DNA damage or replication blocks (1-3). Despite the essential role of ATR in cell cycle signaling and DNA repair processes, little is known about its activation. While there have been no published reports of phosphorylation sites on ATR, Cell Signaling Technology has produced an antibody directed against phospho-ATR (Ser428) that demonstrates in vivo and UV-induced phosphorylation of this protein. This reagent could prove to be a valuable tool for monitoring ATR activation. Proline-directed phosphorylation sites like this one are often targeted by CDKs and MAPKs and can often dramatically affect protein conformation (4,5).

  1. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
  2. Abraham, R.T. (2004) DNA Repair (Amst) 3, 883-887.
  3. Shechter, D. et al. (2004) DNA Repair (Amst) 3, 901-908.
  4. Pinna, L.A. and Ruzzene, M. (1996) Biochim. Biophys. Acta 1314, 191-225.
  5. Zhou, X.Z. et al. (1999) Cell Mol. Life Sci. 56, 788-806.

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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