Western blot analysis of extracts from 293 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® Rictor siRNA I (+), or SignalSilence® Rictor siRNA II #8622 (+), using Rictor (53A2) Rabbit mAb #2114 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Rictor (53A2) Rabbit mAb confirms silencing of rictor expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
CST recommends transfection with 100 nM SignalSilence® Rictor 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® Rictor siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit rictor 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.
Cell growth is a fundamental biological process whereby cells accumulate mass and increase in size. The mammalian TOR (mTOR) pathway regulates growth by coordinating energy and nutrient signals with growth factor-derived signals (1). mTOR is a large protein kinase with two different complexes. One complex contains mTOR, GβL and raptor, which is a target of rapamycin. The other complex, insensitive to rapamycin, includes mTOR, GβL, Sin1, and rictor (1). The mTOR-rictor complex phosphorylates Ser473 of Akt/PKB in vitro (2). This phosphorylation is essential for full Akt/PKB activation. Furthermore, an siRNA knockdown of rictor inhibits Ser473 phosphorylation in 3T3-L1 adipocytes (3). This complex has also been shown to phosphorylate the rapamycin-resistant mutants of S6K1, another effector of mTOR (4).
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SignalSilence is a registered trademark of Cell Signaling Technology, Inc.