Western blot analysis of extracts from NIH/3T3 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® PI3 Kinase p85α siRNA I (Mouse Specific) (+), using PI3 Kinase p85 (19H8) Rabbit mAb #4257 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The PI3 Kinase p85 (19H8) Rabbit mAb confirms silencing of PI3 Kinase p85α expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
CST recommends transfection with 100 nM SignalSilence® PI3 Kinase p85-α siRNA I (Mouse Specific) 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.
SignalSilence® siRNA is supplied in RNAse-free water. Aliquot and store at -20ºC.
SignalSilence® PI3 Kinase p85α siRNA I (Mouse Specific) from Cell Signaling Technology (CST) allows the researcher to specifically inhibit PI3 Kinase p85α 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.
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.
Phosphoinositide 3-kinase (PI3K) catalyzes the production of phosphatidylinositol-3,4,5-triphosphate by phosphorylating phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). Growth factors and hormones trigger this phosphorylation event, which in turn coordinates cell growth, cell cycle entry, cell migration, and cell survival (1). PTEN reverses this process, and research studies have shown that the PI3K signaling pathway is constitutively activated in human cancers that have loss of function of PTEN (2). PI3Ks are composed of a catalytic subunit (p110) and a regulatory subunit. Various isoforms of the catalytic subunit (p110α, p110β, p110γ, and p110δ) have been isolated, and the regulatory subunits that associate with p110α, p110β, and p110δ are p85α and p85β (3). In contrast, p110γ associates with a p101 regulatory subunit that is unrelated to p85. Furthermore, p110γ is activated by βγ subunits of heterotrimeric G proteins (4).
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SignalSilence is a registered trademark of Cell Signaling Technology, Inc.
|6912S||300 µl (3 nmol)||N/A|