Figure 2. northern blot analyses of GEO-D03-expressed mRNAS. (A) Schematic showing reading frames for proteins expressed by GEO-D03 DNA (open rectangles) and size classes of spliced mRNAs (filled rectangles). The asterisks indicate multiple, closely spaced spliced sites. (B) Northern blot probed for GM-CSF sequences. The sequences in each size class of mRNA and the proteins expressed by each size class are shown to the right of the blot. GEO-D03 DNA and pGA2/JS7 were transfected into HEK293T cells in six-well plates at 1 µg DNA per well using the LipofectAmine 2000 transfection reagent (Life Technologies). After a two-day incubation, cells were trypsinized, and RNA was purified from the cells with the RNAqueous kit (Life Technologies) according to the kit protocol. Electrophoresis and transfer were performed using the NorthernMax kit (Life Technologies) according to the kit protocol. The biotinylated Gag probe was hybridized to the membrane and the blot processed according to standard procedures and scanned on an Odyssey infrared scanner (Li-Cor). Once scanned, the blot was stripped with 0.5% SDS and re-probed with the GM-CSF probe.