Cell Signaling Technology

Product Pathways - MAPK Signaling

SignalSilence® MEK1 siRNA Kit (Human Specific) #6425

Kit Includes

SignalSilence® MEK1 siRNA (Human Specific) #6426
pre-validated single sequence siRNA duplex guaranteed to specifically inhibit MEK1 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 MEK1 Antibody #9124
to confirm silencing of MEK1 expression.
Non-targeted GCK Antibody #3782
to control for protein loading and MEK1 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 (lane 1), MEK1 siRNA (lane 2), MEK2 siRNA (lane 3) or MEK1 and MEK2 siRNA (Lane 4), using MEK1 Antibody #9124 (upper) or MEK2 Antibody #9125 (lower) in combination with GCK Antibody #3782. The MEK1 Antibody confirms silencing of MEK1 expression, MEK2 Antibody demonstrates MEK1 siRNA has no effect on its homologue MEK2, and GCK Antibody is used to control for loading and specificity of MEK1 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® MEK1 siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit MEK1 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® MEK1 siRNA Kit includes MEK1 siRNA, a target-specific MEK1 Antibody to confirm the silencing of MEK1 expression and a non-target antibody (GCK) to control for loading and monitor specificity of MEK1 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 MEK1 siRNA 48 to 72 hours prior to cell lysis. See Protocol for transfection procedure.

Tested Cell Lines

HeLa

Background

MEK1 and MEK2, also called MAPK or Erk kinases, are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation (1-3). Activation of MEK1 and MEK2 occurs through phosphorylation of two serine residues at positions 217 and 221 (in the activation loop of subdomain VIII) by Raf-like molecules. MEK1/2 is activated by a wide variety of growth factors and cytokines and also by membrane depolarization and calcium influx (1-4). Constitutively active forms of MEK1/2 are sufficient for the transformation of NIH/3T3 cells or the differentiation of PC12 cells (4). MEK activates p44 and p42 MAP kinase by phosphorylating both threonine and tyrosine residues at sites located within the activation loop of kinase subdomain VIII.

Small interfering RNA (siRNA) has been used to specifically silence MEK1 expression in HEK293T cells (5).

  1. Crews, C.M. et al. (1992) Science 258, 478-480.
  2. Alessi, D.R. et al. (1994) EMBO J. 13, 1610-1619.
  3. Rosen, L.B. et al. (1994) Neuron 12, 1207-1221.
  4. Cowley, S. et al. (1994) Cell 77, 841-852.
  5. 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.

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