Western blot analysis of extracts from HeLa cells, transfected with either 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-), SignalSilence® PKA C-α siRNA II (+) or SignalSilence® PKA C-α siRNA I #6406 (+), using PKA C-α Antibody #4782 and α-Tubulin (11H10) Rabbit mAb #2125. The PKA C-α antibody confirms silencing of PKA C-α expression and α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of PKA C-α siRNA.
CST recommends transfection with 100 nM PKA C-α siRNA 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® PKA C-α siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit PKA C-α 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 are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
The second messenger cyclic AMP (cAMP) activates cAMP-dependent protein kinase (PKA or cAPK) in mammalian cells and controls many cellular mechanisms such as gene transcription, ion transport, and protein phosphorylation (1). Inactive PKA is a heterotetramer composed of a regulatory subunit (R) dimer and a catalytic subunit (C) dimer. In this inactive state, the pseudosubstrate sequences on the R subunits block the active sites on the C subunits. Three C subunit isoforms (C-α, C-β, and C-γ) and two families of regulatory subunits (RI and RII) with distinct cAMP binding properties have been identified. The two R families exist in two isoforms, α and β (RI-α, RI-β, RII-α, and RII-β). Upon binding of cAMP to the R subunits, the autoinhibitory contact is eased and active monomeric C subunits are released. PKA shares substrate specificity with Akt (PKB) and PKC, which are characterized by an arginine at position -3 relative to the phosphorylated serine or threonine residue (2). Substrates that present this consensus sequence and have been shown to be phosphorylated by PKA are Bad (Ser155), CREB (Ser133), and GSK-3 (GSK-3α Ser21 and GSK-3β Ser9) (3-5). In addition, combined knock-down of PKA C-α and -β blocks cAMP-mediated phosphorylation of Raf (Ser43 and Ser259) (6). Autophosphorylation and phosphorylation by PDK-1 are two known mechanisms responsible for phosphorylation of the C subunit at Thr197 (7).
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