Cell Signaling Technology

Product Pathways - Apoptosis

SignalSilence® LC3B siRNA I #6212

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 (-), SignalSilence® LC3B siRNA I (+) or SignalSilence® LC3B siRNA II #6213 (+), using LC3B (D11) XP® Rabbit mAb #3868 and α-Tubulin (11H10) Rabbit mAb #2125. The LC3B (D11) XP® Rabbit mAb confirms silencing of LC3B expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of LC3B siRNA.

Description

SignalSilence® LC3B siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit LC3B expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. LC3B siRNA I will not inhibit expression of LC3A or LC3C. 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 LC3B 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

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation, but it has also been associated with a number of physiological processes including development, differentiation, neurodegenerative diseases, infection, and cancer (3). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (4) and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 critical for autophagy (5). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications during autophagy (6-9). Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles (6-10). The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form, LC3-II, have been used as indicators of autophagy (11).

  1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot. Cell 1, 11-21.
  2. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ. 12 Suppl 2, 1509-1518.
  3. Levine, B. and Yuan, J. (2005) J. Clin. Invest. 115, 2679-2688.
  4. Mann, S.S. and Hammarback, J.A. (1994) J. Biol. Chem. 269, 11492-11497.
  5. Lang, T. et al. (1998) EMBO J. 17, 3597-3607.
  6. Kabeya, Y. et al. (2000) EMBO J. 19, 5720-5728.
  7. He, H. et al. (2003) J. Biol. Chem. 278, 29278-29287.
  8. Tanida, I. et al. (2004) J. Biol. Chem. 279, 47704-47710.
  9. Wu, J. et al. (2006) Biochem. Biophys. Res. Commun. 339, 437-442.
  10. Ichimura, Y. et al. (2000) Nature 408, 488-492.
  11. Kabeya, Y. et al. (2004) J. Cell Sci. 117, 2805-2812.

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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