Western blot analysis of extracts from MCF7 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® Hexokinase II siRNA I (+), using Hexokinase II (C64G5) Rabbit mAb #2867 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The Hexokinase II (C64G5) Rabbit mAb confirms silencing of hexokinase II expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.
CST recommends transfection with 100 nM SignalSilence® Hexokinase II 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.
Hexokinase II siRNA I is supplied in RNase-free water. Aliquot and store at -20ºC.
SignalSilence® Hexokinase II siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit hexokinase II 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.
Hexokinase catalyzes the conversion of glucose to glucose-6-phosphate, the first step in glycolysis. Four distinct mammalian hexokinase isoforms, designated as hexokinase I, II, III, and IV (glucokinase), have been identified. Hexokinases I, II, and III are associated with the outer mitochondrial membrane and are critical for maintaining an elevated rate of aerobic glycolysis in cancer cells (Warburg Effect) (1) in order to compensate for the increased energy demands associated with rapid cell growth and proliferation (2,3).
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