Western blot analysis of extracts from RD cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® TNF-R1 siRNA I (+), using TNF-R1 (C25C1) Rabbit mAb #3736 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The TNF-R1 (C25C1) Rabbit mAb confirms silencing of TNF-R1 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.Learn more about how we get our images
CST recommends transfection with 100 nM SignalSilence® TNF-R1 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® TNF-R1 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit TNF-R1 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.
TNF-α is an important cytokine produced by numerous cell types including neutrophils, activated lymphoctyes, macrophages and NK cells. It plays a critical role in inflammatory responses and in apoptosis (1). TNF-α exists as a membrane-anchored and soluble form, both of which show biological activity. Response to TNF-α is mediated through two receptors, TNF-R1, which is widely expressed, and TNF-R2, which is expressed mainly in immune and endothelial cells (2). Antagonists to TNF-α have been validated as therapeutic targets for rheumatoid arthritis and other immune disorders (3).
The two receptors for TNF-α, TNF-R1 (55 kDa) and TNF-R2 (75 kDa) can mediate distinct cellular responses (4,5). In most cases cytotoxicity elicited by TNF-α has been reported to act through TNF-R1 (6,7). Cytotoxicity is mediated by a "death domain" within the intracellular region of the receptor that binds to the death domain adaptor protein TRADD and triggers the activation of caspases (8). Soluble forms of both receptors have also been characterized which can bind TNF-α and may play an important role in immune disorders (9,10).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. SignalSilence is a registered trademark of Cell Signaling Technology, Inc.
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