Cell Signaling Technology

Product Pathways - MAPK Signaling

SignalSilence® p44 MAPK siRNA Kit (Human Specific) #6435

Kit Includes

SignalSilence® p44 MAPK siRNA #6436
pre-validated single sequence siRNA duplex guaranteed to specifically inhibit p44 MAPK 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 MAPK Antibody #9102
to confirm silencing of p44 MAPK expression.
Non-targeted GCK Antibody #3782
to control for protein loading and p44 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 100 nM control siRNA #6201 (-) or p44 MAPK siRNA (+), using p44/42 MAP Kinase Antibody #9102 and GCK Antibody #3782. The p44 MAP Kinase Antibody confirms silencing of p44 MAPK expression, and GCK Antibody is used to control for loading and specificity of p44 MAPK siRNA.

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® p44 MAPK siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit p44 MAPK expression in human cell lines. 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. All SignalSilence® siRNA kits are rigorously tested in-house and have been shown to reduce protein expression. SignalSilence® p44 MAPK siRNA Kit includes p44 MAPK siRNA, a target-specific p44/42 MAP Kinase Antibody to confirm the silencing of p44 MAPK expression and a non-target antibody (GCK) to control for loading and monitor specificity of p44 MAPK siRNA. Additionally, a fluorescent tagged non-targeted siRNA control allows the user to monitor transfection efficiency and siRNA specificity. The kit also includes a transfection reagent designed solely for efficient siRNA delivery in mammalian cells.

Directions for Use

CST recommends transfection with 100 nM human-specific p44 MAPK 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 or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family as well as Mos and Tpl2/Cot. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 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.

Small interfering RNA (siRNA) has been used to specifically silence p44 MAP Kianse expression in HEK293T cells (7).

  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.
  11. Castro-Obregon, S. et al. (2004) J. Biol. Chem. In press, .

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.

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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