Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® CDK2 siRNA I (+) or SignalSilence® CDK2 siRNA II #7417 (+), using CDK2 (78B2) Rabbit mAb #2546 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The CDK2 (78B2) Rabbit mAb confirms silencing of CDK2 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
CST recommends transfection with 100 nM CDK2 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® CDK2 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit CDK2 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.
Cyclin-dependent kinase 2 (p33CDK2) is an important component of the cell cycle machinery. Like p34cdc2, kinase activity is regulated by phosphorylation state as well as association with a cyclin subunit and a CDK inhibitor. Inhibitory phosphorylation occurs on Thr14 and Tyr15 (1). Inhibition of CDK2-cyclin complexes can also be attributed to association with p27 Kip1 and p21 Waf1/Cip1 (2). Activation of CDK2 complexes requires dephosphorylation of Thr14 and Tyr15 by cdc25 phosphatase and phosphorylation of Thr160 (3), which is mediated by CAK, a complex of CDK7 and cyclin H (4). CDK2/cyclin E kinase activity is important for the G1 to S transition and phosphorylation of the Rb protein. During S-phase, active CDK2/cyclin A complexes predominate and phosphorylate E2F and the active CDK2 complex persists in the nucleus throughout G2 (5).
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