Cell Signaling Technology

Product Pathways - Apoptosis

SignalSilence® Apaf-1 siRNA I #6229

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 (-) or SignalSilence® Apaf-1 siRNA I (+), using Apaf-1 (D5C3) Rabbit mAb #8969 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Apaf-1 (D5C3) Rabbit mAb confirms silencing of Apaf-1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Apaf-1 siRNA I (+), using Apaf-1 (D7G4) Rabbit mAb #8723 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Apaf-1 (D7G4) Rabbit mAb confirms silencing of Apaf-1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

SignalSilence® Apaf-1 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Apaf-1 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 Apaf-1 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

Apoptotic protease activating factor 1 (Apaf-1), originally identified as the mammalian homolog of the C. elegans apoptotic regulatory protein CED-4, is an important signaling protein involved in the activation of caspase-9 during apoptosis (1). Cytosolic Apaf-1 forms a complex with caspase-9 in the presence of cytochrome c and dATP, ultimately leading to caspase-9 activation and subsequent activation of caspase-3 (2,3). The protein contains an amino-terminal CARD domain, a central CED-4 homology domain, and multiple WD-40 repeats at the carboxy-terminus. Several isoforms of Apaf-1 are expressed through alternative splicing generating a small insert following the CARD domain as well as an extra WD-40 repeat (4). Apaf-1 knock-out mice display widespread defects in apoptosis and resistance to a variety of apoptotic stimuli (5,6).

  1. Zou, H. et al. (1997) Cell 90, 405-13.
  2. Zou, H. et al. (1999) J Biol Chem 274, 11549-56.
  3. Saleh, A. et al. (1999) J Biol Chem 274, 17941-5.
  4. Benedict, M.A. et al. (2000) J Biol Chem 275, 8461-8.
  5. Cecconi, F. et al. (1998) Cell 94, 727-37.
  6. Yoshida, H. et al. (1998) Cell 94, 739-50.

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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