Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® Skp2 siRNA I (+), or SignalSilence® Skp2 siRNA II #7756 (+), using Skp2 (L70) Antibody #4313 (upper) or GAPDH (14C10) Rabbit mAb #2118 (lower). The Skp2 (L70) Antibody confirms silencing of Skp2 expression, while the GAPDH (14C10) Rabbit mAb is used as a loading control.
CST recommends transfection with 100 nM SignalSilence® Skp2 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.
SignalSilence® siRNA is supplied in RNAse-free water. Aliquot and store at -20ºC.
SignalSilence® Skp2 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Skp2 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.
Members of the F-box family of proteins are characterized by the approximate 40 amino acid F-box motif named after cyclin F (1,2). F-box proteins constitute one of the four subunits of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex. The substrate specificity of SCF complexes is determined by the interchangeable F-box proteins, which act as adaptors by associating with phosphorylated substrate proteins and recruiting them to the SCF core. F-box proteins contain two fundamental domains: the F-box motif mediates binding to Skp1 and a leucine rich repeat (LRR) domain mediates substrate interactions.
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