Cell Signaling Technology

Product Pathways - Ca / cAMP / Lipid Signaling

SignalSilence® PKA C-α siRNA Kit #6405

Kit Includes

SignalSilence® PKA C-alpha siRNA #6406
pre-validated single sequence siRNA duplex guaranteed to specifically inhibit PKA C-alpha 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 PKA C-alpha Antibody #4782
to confirm silencing of PKA C-alpha expression.
Non-targeted Akt1 (2H10) Monoclonal Antibody #2967
to control for protein loading and PKA C-alpha siRNA specificity.
*Transfection Reagent
designed specifically for highly efficient siRNA delivery in mammalian cells. Transfection reagent is supplied by Mirus.

*Store at 4°C (packaged separately)

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells transfected with non-targeted (-) or PKA C-alpha (+) siRNA. PKA C-alpha was detected using the PKA C-alpha Antibody #4782, and Akt1 was detected using the Akt1 (2H10) Monoclonal Antibody #2967. The PKA C-alpha Antibody confirms silencing of PKA C-alpha expression, and the Akt1 Antibody is used to control for loading and specificity of PKA C-alpha 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® PKA C-alpha siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit PKA C-alpha expression. 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® PKA C-alpha siRNA Kit includes PKA C-alpha siRNA, a target-specific PKA C-alpha Antibody to confirm the silencing of PKA C-alpha expression and a non-target antibody (Akt) to control for loading and monitor specificity of PKA C-alpha siRNA. Additionally, a fluorescein-labeled non-targeted siRNA control allows the user to monitor transfection efficiency and siRNA specificity. The kit also contains a transfection reagent designed solely for efficient siRNA delivery in mammalian cells.

Directions for Use

CST recommends transfection with 100 nM SignalSilence® PKA C-alpha siRNA 48 hours prior to cell lysis. See protocol for transfection procedure.

Background

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, and 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. Within the two R families, two isoforms, α and β (RI-α, RI-β, RII-α and RII-β) exist. 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 is 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 C-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).

Small interfering RNA (siRNA) has been used to specifically silence PKA C-alpha in NIH/3T3 cells (6).

  1. Montminy, M. (1997) Annu. Rev. Biochem. 66, 807-822.
  2. Dell'Acqua, M.L. and Scott, J.D. (1997) J. Biol. Chem. 272, 12881-12884.
  3. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
  4. Gonzalez, G.A. and Montminy, M.R. (1989) Cell 59, 675-680.
  5. Fang, X. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 11960-11965.
  6. Dumaz, N. and Marais, R. (2003) J. Biol. Chem. 278, 29819 -29823.
  7. Moore, M.J. et al. (2002) J. Biol. Chem. 277, 47878-47884.

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.

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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