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 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.
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.
SignalSilence® siRNA is supplied in RNAse-free water. Aliquot and store at -20ºC.
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.
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.
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. ATR was long thought to exist in a constitutively active state in cells, with DNA damage-induced signaling occurring via recruitment of ATR to single stranded DNA and sites of replication stress. Phosphorylation of ATR at serine 428 in response to UV-induced DNA damage has been suggested as a means of activating ATR (4,5). Recent work has shown autophosphorylation of ATR at threonine 1989. Like ATM Ser1981, phosphorylation of ATR Thr1989 occurs in response to DNA damage, indicating that phosphorylation at this site is important in ATR-mediated signaling (6,7).
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