Cell Signaling Technology

Product Pathways - MAPK Signaling

SignalSilence® MKK3 siRNA I #6294

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® MKK3 siRNA I (+) or SignalSilence® MKK3 siRNA II #6295 (+), using MKK3 Antibody #9232 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The MKK3 Antibody confirms silencing of MKK3 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

SignalSilence® MKK3 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit MKK3 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 MKK3 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

MKK3 and MKK6 are two closely related dual-specificity protein kinases that activate p38 MAP kinase (1-5). MKK3 and MKK6 both phosphorylate and activate p38 MAP kinase at its activation site, Thr-Gly-Tyr, but do not phosphorylate or activate Erk1/2 or SAPK/JNK. Phosphorylation of p38 MAP kinase dramatically stimulates its ability to phosphorylate protein substrates such as ATF-2 and Elk-1. MKK3 and MKK6 are both activated by different forms of cellular stress and inflammatory cytokines (4,5). Activation of MKK3 and MKK6 occurs through phosphorylation at Ser189 and Thr222 on MKK3 (2) and Ser207 and Thr211 on MKK6 (4,5).

  1. Derijard, B. et al. (1995) Science 267, 682-685.
  2. Raingeaud, J. et al. (1995) J. Biol. Chem. 270, 7420-7426.
  3. Sluss, H.K. et al. (1994) Mol. Cell. Biol. 14, 8376-8384.
  4. Raingeaud, J. et al. (1996) Mol. Cell. Biol. 16(3), 1247-1255.
  5. Han, J. et al. (1996) J. Biol. Chem. 271, 2886-2891.

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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