Cell Signaling Technology

Product Pathways - Cytoskeletal Signaling

SignalSilence® Rabex-5 siRNA I #6437

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 SH-SY5Y cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Rabex-5 siRNA I (+), using Rabex-5 (D21F12) Rabbit mAb #7622 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Rabex-5 (D21F12) Rabbit mAb confirms silencing of Rabex-5 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

SignalSilence® Rabex-5 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Rabex-5 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 Rabex-5 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

Rabex-5, also called RabGEF1 and RAP1, was identified as a guanine nucleotide exchange factor (GEF) for Rab5, a member of the Ras superfamily of small Rab GTPases (1). Rabex-5 generates the GTP-bound active form of Rab5 and forms a tight association with its effector protein Rabaptin-5 (2). This complex localizes to endosomal membranes where it functions as a key regulator of vesicular trafficking during early endocytosis (3,4). Rabex-5 is also monoubiquitinated and has ubiquitin ligase activity that regulates its recruitment to early endosomes (5,6). The conformational change between Rab5 GTP/GDP states is essential for its biological function as a rate limiting regulator at multiple steps during endocytosis (5). Through its control of endosomal trafficking and endocytosis, Rabex-5 has been shown to negatively regulate NGF-mediated neurite outgrowth (7) as well as FcεRI-dependent mast cell activation (8).

  1. Horiuchi, H. et al. (1997) Cell 90, 1149-59.
  2. Lippé, R. et al. (2001) Mol Biol Cell 12, 2219-28.
  3. Zerial, M. and McBride, H. (2001) Nat Rev Mol Cell Biol 2, 107-17.
  4. van der Bliek, A.M. (2005) Nat Cell Biol 7, 548-50.
  5. Mattera, R. et al. (2006) J Biol Chem 281, 6874-83.
  6. Mattera, R. and Bonifacino, J.S. (2008) EMBO J 27, 2484-94.
  7. Liu, J. et al. (2007) Mol Biol Cell 18, 1375-84.
  8. Tam, S.Y. et al. (2004) Nat Immunol 5, 844-52.

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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