Cell Signaling Technology

Product Pathways - Ca / cAMP / Lipid Signaling

SignalSilence® PLCγ1 siRNA I #6293

Applications Reactivity
Transfection H

Reactivity Key:  H=Human
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® PLCγ1 siRNA I (+) or SignalSilence®PLCγ1 siRNA II #6254 (+), using PLCγ1 (D9H10) XP® Rabbit mAb #5690 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The PLCγ1 (D9H10) XP® Rabbit mAb confirms silencing of PLCγ1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

SignalSilence® PLCγ1 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit PLCγ1 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.

Quality Control

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 PLCγ1 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.

Background

Phosphoinositide-specific phospholipase C (PLC) plays a significant role in transmembrane signaling. In response to extracellular stimuli such as hormones, growth factors, and neurotransmitters, PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two secondary messengers: inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (1). At least four families of PLCs have been identified: PLCβ, PLCγ, PLCδ, and PLCε. Phosphorylation is one of the key mechanisms that regulate the activity of PLC. PLCγ is activated by both receptor and non-receptor tyrosine kinases (2). PLCγ forms a complex with EGF and PDGF receptors, which leads to the phosphorylation of PLCγ at Tyr771, 783, and 1245 (3). Phosphorylation by Syk at Tyr783 activates the enzymatic activity of PLCγ1 (4). PLCγ2 is engaged in antigen-dependent signaling in B cells and collagen-dependent signaling in platelets. Phosphorylation by Btk or Lck at Tyr753, 759, 1197, and 1217 is correlated with PLCγ2 activity (5,6).

  1. Singer, W.D. et al. (1997) Annu Rev Biochem 66, 475-509.
  2. Margolis, B. et al. (1989) Cell 57, 1101-7.
  3. Kim, H.K. et al. (1991) Cell 65, 435-41.
  4. Wang, Z. et al. (1998) Mol Cell Biol 18, 590-7.
  5. Watanabe, D. et al. (2001) J Biol Chem 276, 38595-601.
  6. Ozdener, F. et al. (2002) Mol Pharmacol 62, 672-9.

Application References

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Companion Products

Limited Use Label License, RNA interference: This product is licensed under European Patent 1144623 and foreign equivalents from Ribopharma AG, Kulmbach, Germany and is provided only for use in non-commercial research specifically excluding use (a) in drug discovery or drug development, including target identification or target validation, by or on behalf of a commercial entity, (b) for contract research or commercial screening services, (c) for the production or manufacture of siRNA-related products for sale, or (d) for the generation of commercial databases for sale to Third Parties. Information about licenses for these and other commercial uses is available from Ribopharma AG, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Germany.

For Research Use Only. Not For Use In Diagnostic Procedures.

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