Fluorescent detection of SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 in living HeLa cells 24 hours post-transfection, demonstrating nearly 100% transfection efficiency.
Western blot analysis of extracts from 293 and Hela cells, transfected with control (-) or p21 Waf1/Cip1 (+) siRNA. p21 Waf1/Cip1 was detected using p21 Waf1/Cip1 (DCS60) Mouse Monoclonal Antibody #2946, p42 MAPK was detected using p42 MAPK (3A7) Mouse Monoclonal Antibody #9107. The p21 Waf1/Cip1 monoclonal antibody confirms silencing of p21 expression, and the p42 monoclonal antibody is used to control for loading and specificity of p21 Waf1/Cip1 siRNA.
CST recommends transfection with 100 nM p21 Waf1/Cip1 siRNA 48 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.
SignalSilence® siRNA is supplied in RNAse-free water. Aliquot and store at -20ºC.
SignalSilence® p21 Waf1/Cip1 siRNA (Human Specific) allows the researcher to specifically inhibit p21 Waf1/Cip1 expression using 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 products from Cell Signaling Technology are rigorously tested in-house and have been shown to reduce protein expression in specified cell lines.
The tumor suppressor protein p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression. It functions in stoichiometric relationships forming heterotrimeric complexes with cyclins and cyclin-dependent kinases. In association with CDK2 complexes, it serves to inhibit kinase activity and block progression through G1/S (1). However, p21 may also enhance assembly and activity in complexes of CDK4 or CDK6 and cyclin D (2). The carboxy-terminal region of p21 is sufficient to bind and inhibit PCNA, a subunit of DNA polymerase, and may coordinate DNA replication with cell cycle progression (3). Upon UV damage or during cell cycle stages when cdc2/cyclin B or CDK2/cyclin A are active, p53 is phosphorylated and upregulates p21 transcription via a p53-responsive element (4). Protein levels of p21 are downregulated through ubiquitination and proteasomal degradation (5).
RNA interference has been used to silence p21 Cip1 expression in human endothelial cells, preventing TNF-alpha mediated growth inhibition (6).
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