Product Pathways - Apoptosis
SignalSilence® Atg4B siRNA I #6336
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg4B siRNA I (+), using Atg4B Antibody #5299 (upper) or β-Tubulin (9F3) Rabbit mAb #2128 (lower). The Atg4B Antibody confirms silencing of Atg4B expression, while the β-Tubulin (9F3) Rabbit mAb is used as a loading control.
SignalSilence® Atg4B siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Atg4B 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.
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 Atg4B 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.
Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents. Control of autophagy was largely discovered in yeast and involves proteins encoded by a set of autophagy-related genes (Atg) (1). Formation of autophagic vesicles requires a pair of essential ubiquitin-like conjugation systems, Atg12-Atg5 and Atg8-phosphatidylethanolamine (Atg8-PE), which are widely conserved in eukaryotes (2). Numerous mammalian counterparts to yeast Atg proteins have been described, including three Atg8 proteins (GATE-16, GABARAP, and LC3) and four Atg4 homologues (Atg4A/autophagin-2, Atg4B/autophagin-1, Atg4C/autophagin-3, and Atg4D/autophagin-4) (3-5). The cysteine protease Atg4 is pivotal to autophagosome membrane generation and regulation. Atg4 primes the Atg8 homologue for lipidation by cleaving its carboxy terminus and exposing its glycine residue for E1-like enzyme Atg7. The Atg8 homologue is transferred to the E2-like enzyme Atg3 before forming the Atg8-PE conjugate. During later stages of autophagy, Atg4 can reverse this lipidation event by cleaving PE, thereby recycling the Atg8 homologue (6).
- Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
- Ohsumi, Y. (2001) Nat Rev Mol Cell Biol 2, 211-6.
- Kabeya, Y. et al. (2000) EMBO J 19, 5720-8.
- Kabeya, Y. et al. (2004) J Cell Sci 117, 2805-12.
- Mariño, G. et al. (2003) J Biol Chem 278, 3671-8.
- Sou, Y.S. et al. (2008) Mol Biol Cell 19, 4762-75.
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- 6568 SignalSilence® Control siRNA (Unconjugated)
- 6201 SignalSilence® Control siRNA (Fluorescein Conjugate)
- 5299 Atg4B Antibody
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.