Notes: The presence of methyl-cytosine binding domain (MBD) containing proteins and ability to remodel methylated chromatin was investigate in sponges. Specifically, the sponge MBD2 and GATAD2A protein interaction was monitored in cells using the NanoBRET™ Protein-Protein Interaction System. Multiple coiled-coil fusion constructs were tested to determine optimal signal intensity. Interestingly, while DNA methylation was observed in sponges, this interaction was much lower affinity than in vertebrate organisms. (5057)

Notes: The T7 RiboMAX™ Express RNAi System was used to produce double-stranded RNAs based on the sequence of the Drosophilia mRNA nuclear factor, Nxf1.  The researchers describe producing and annealing two single-stranded RNAs to produce  double-stranded RNA. Schneider S2 cells were incubated with the double-stranded RNA for 45 minutes to 1 hour to knockdown Nxf1 expression.  Expression levels were assessed by Western blotting for the Nxf1 protein and Nxf1 mRNA in situ hybridization experiments on fixed cells.  (3249)

Notes: The T7 RiboMAX™ Express RNAi System was used to create shRNAs to mouse and human neuron-restrictive silencer factor (NRSF). A scrambled sequence shRNA was also created with the system and used as a control. One to three micrograms of each shRNA were in transfecting NS20Y and HeLa cells.  The siRNA Target Designer (http://www.promega.com/siRNADesigner/) was used to design an siRNA against NRSF. (3208)

Notes: In this study, siRNA was used to reduce the level of the PU.1 transcription factor.  The siRNA was generated using the T7 RiboMAX™ Express RNAi System with design assistance from the siRNA Target Designer (http://www.promega.com/siRNADesigner/). Annealed siRNA was purified by isopropanol precipitation. Forty-eight hours after transfecting 2.5ug of siRNA into RAW264.7 cells, RNA and protein were isolated from the cells and the siRNA effect was analyzed by RT-PCR and Western blot. (3062)