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By Kennedy R.J.

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FACS analysis is performed on a Beckon Dickinson FACS Caliber machine with a 488-nm laser. 8. Mean EGFP is the average emission in relative fluorescence units (RFU) of approx 10,000 cells that reside within a gated value set at a threshold above background fluorescence. 5. Measuring Exon IIIb Inclusion Using pGIIIb Plasmids The plasmid pEGFPN1 (Clontech, Palo Alto, CA), which can drive EGFP expression in both DT3 and AT3 cells, was used to construct the pG family of alternative splicing reporter vectors.

Several factors have been implicated in mediating IIIb silencing; the polypyrimidine tract binding protein (PTB) binds the ISS elements that flank IIIb and silences this exon (11). HnRNP A1 has also been implicated in IIIb silencing. Artificial recruitment of hnRNP A1 protein to IIIb leads to decreased IIIb inclusion (18). IIIb inclusion is likely mediated by an epithelial specific factor, which we posit interacts with and stabilizes a secondary structure predicted to be formed by ISAR-IAS2 base pairing (7,9).

Change the media every 3 d during this incubation. Trypsinize the flask, then replate into a T75 flask in the same selective media (see Note 14). After sufficient numbers of blasticidin/hygromycin resistant cells have been produced, the cells are ready for flow cytometry analysis. 4. 5. 6. 3. Fluorescent Microscopy 1. Cells are plated at 3 × 105/well in a six-well plate, where each well contains a sterile glass coverslip. 2. The following day, remove the media and wash the cells once with 2 mL PBS.

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A Refinement of the Michelson-Morley Experiment by Kennedy R.J.

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