Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-), SignalSilence® Rb siRNA I #6451 (+) or SignalSilence® Rb siRNA II (+), using Rb (4H1) Mouse mAb #9309 and α-Tubulin (11H10) Rabbit mAb #2125. The Rb (4H1) Mouse mAb confirms silencing of Rb expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of Rb siRNA.
CST recommends transfection with 100 nM Rb siRNA II 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® Rb siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Rb 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 are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
The retinoblastoma tumor suppressor protein Rb regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).
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