|6578||SignalSilence® p42 MAP Kinase (Erk2) siRNA II||
Fluorescent detection of SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 in living HeLa cells 24 hours post-transfection, demonstrating nearly 100% transfection efficiency.
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 specifically that the pool p42 MAPK siRNA does not interfere with the expression of p44 MAPK, p38 MAPK or SAPK/JNKs.
CST recommends transfection with 5 to 20 nM pool p42 MAP Kinase siRNA 48 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.
SignalSilence® siRNA is supplied in RNAse-free water. Aliquot and store at -20ºC.
SignalSilence® Pool p42 MAP Kinase siRNA from Cell Signaling Technology allows the researcher to specifically inhibit p42 MAP Kinase expression using 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 products are rigorously tested in-house and have been shown to reduce protein expression in specified cell lines.
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 research investigators consider it 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.
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