Product Pathways - Ca / cAMP / Lipid Signaling
SignalSilence® PKCζ siRNA II #8939
|8939S||300 µl (3 nmol)||---||In Stock||---|
|8939||carrier free and custom formulation / quantity||email request|
Product is assembled upon order. Please allow up to three business days for your product to be processed.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® PKCζ siRNA I #8672 (+), or SignalSilence® PKCζ siRNA II (+), using PKCζ (C24E6) Rabbit mAb #9368 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The PKCζ (C24E6) Rabbit mAb confirms silencing of PKCζ expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
SignalSilence® PKCζ siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit PKCζ expression by 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.
Directions For Use
CST recommends transfection with 100 nM SignalSilence® PKCζ siRNA II 48 to 72 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.
Activation of protein kinase C (PKC) is one of the earliest events in a cascade that controls a variety of cellular responses, including secretion, gene expression, proliferation, and muscle contraction (1,2). PKC isoforms belong to three groups based on calcium dependency and activators. Classical PKCs are calcium-dependent via their C2 domains and are activated by phosphatidylserine (PS), diacylglycerol (DAG), and phorbol esters (TPA, PMA) through their cysteine-rich C1 domains. Both novel and atypical PKCs are calcium-independent, but only novel PKCs are activated by PS, DAG, and phorbol esters (3-5). Members of these three PKC groups contain a pseudo-substrate or autoinhibitory domain that binds to substrate-binding sites in the catalytic domain to prevent activation in the absence of cofactors or activators. Control of PKC activity is regulated through three distinct phosphorylation events. Phosphorylation occurs in vivo at Thr500 in the activation loop, at Thr641 through autophosphorylation, and at the carboxy-terminal hydrophobic site Ser660 (2). Atypical PKC isoforms lack hydrophobic region phosphorylation, which correlates with the presence of glutamic acid rather than the serine or threonine residues found in more typical PKC isoforms. The enzyme PDK1 or a close relative is responsible for PKC activation. A recent addition to the PKC superfamily is PKCμ (PKD), which is regulated by DAG and TPA through its C1 domain. PKD is distinguished by the presence of a PH domain and by its unique substrate recognition and Golgi localization (6). PKC-related kinases (PRK) lack the C1 domain and do not respond to DAG or phorbol esters. Phosphatidylinositol lipids activate PRKs, and small Rho-family GTPases bind to the homology region 1 (HR1) to regulate PRK kinase activity (7).
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- Moscat, J. and Diaz-Meco, M.T. (2000) EMBO Rep 1, 399-403.
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- Flynn, P. et al. (2000) J Biol Chem 275, 11064-70.
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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
SignalSilence is a registered trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.