Cell Signaling Technology

Product Pathways - MAPK Signaling

SignalSilence® Pool p42 MAPK siRNA Kit #6390

Kit Includes

SignalSilence® Pool p42 MAP Kinase (Erk2) siRNA #6391
pre-validated heterogeneous duplex siRNA guaranteed to specifically inhibit p42 expression in specified cell lines.
SignalSilence® Control siRNA (Fluorescein Conjugate) #6201
a non-targeted negative control duplex designed to monitor transfection efficiency using microscopy and to monitor siRNA specificity.Kit contains enough for 4(S) or 10 (L) transfections.
Targeted p44/42 MAP Kinase Antibody #9102
to confirm silencing of p42 MAP Kinase (Erk2) expression.
Non-targeted ATP-Citrate Lyase Antibody #4332
to control for protein loading and Pool p42 MAPK siRNA specificity.
*Transfection Reagent
designed specifically for highly efficient siRNA delivery in mammalian cells. Transfection reagent is supplied by Mirus.

*Store at 4° (packaged separately)

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells transfected with 20 nM control siRNA #6201 (-) or 2, 5, or 20 nM pool 42 MAPK siRNA, using p44/42 MAP Kinase Antibody #9102 (upper), p38 MAP Kinase Antibody #9212 (middle) and SAPK/JNK (56G8) Rabbit mAb #9258 (lower) in combination with ATP-Citrate Lyase Antibody #4332. The 44/42 MAP Kinase Antibody confirms silencing of p42 MAP Kinase expression, and ATP-Citrate Lyase Antibody is used to control for loading and specificity of pool p42 MAP Kinase siRNA. The experiment also demonstrates that the pool p42 MAPK siRNA does not interfere with the expression of p44 MAPK, p38 MAPK or SAPK/JNKs.

Fluorescent Detection

Fluorescent Detection

Fluorescent detection of SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 in living HeLa cells 24 hours post-transfection, demonstrating nearly 100% transfection efficiency.

Description

SignalSilence® Pool p42 MAP Kinase siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit p42 MAP Kinase expression. The kit utilizes RNA interference, a method in which gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. The p42 MAP Kinase siRNA is a hetergeneous mixture of 21-22 bp siRNAs. A 283 bp cDNA template of human p42 MAP Kinase was transcribed by T7 RNA polymerase to create double-stranded RNA (dsRNA). RNase III was used to cleave the dsRNA in the presence of Mn2+ buffer to 21-22 bp siRNA. All SignalSilence® siRNA kits are rigorously tested in-house and have been shown to reduce protein expression in specified cell lines. SignalSilence® Pool p42 MAPK siRNA Kit includes Pool p42 MAP Kinase siRNA, a target-specific p44/42 MAP Kinase Antibody to confirm the silencing of p42 MAP Kinase expression and a non-target antibody (ATP-Citrate Lyase) to control for loading and monitor specificity of p42 MAP Kinase siRNA. Additionally, a fluorescein-labeled non-targeted siRNA control allows the user to monitor transfection efficiency and siRNA specificity. The kit also contains a transfection reagent designed solely for efficient siRNA delivery in mammalian cells.

Directions for Use

CST recommends transfection with 5 to 20 nM pool p42 MAP Kinase siRNA 48 to 72 hours prior to cell lysis. See Protocol for transfection procedure.

Background

Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (ERK1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3) and is an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase. While multiple ERK1/2 MAP3Ks have been identified, including the Raf family, Mos, and Tpl2/Cot, MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate ERK1/p44 and ERK2/p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of ERK1/2 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). ERK1/2 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors such as U0126 and PD98059.

  1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
  2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.
  3. Meloche, S. and Pouyssegur, J. (2007) Oncogene 26, 3227-39.
  4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
  5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
  6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
  7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
  8. Marais, R. et al. (1993) Cell 73, 381-93.
  9. Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
  10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.

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.

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